Method for assaying the sod activity by using a self-oxidizable compound necessary for its implementation, self-oxidizable compounds and preparation thereof

ABSTRACT

The method for assaying the SOD (super oxide dismutase) activity in liquid medium is based on the activation of self-oxidization, by SOD activity, of a reactive agent having the general formula (I) wherein either n is 1 or 2, R 1  is --OR 4  or --NR 5  R 6  ; R 2  is H, --OR 4 , alkyl (1-6C), --CH 2  -- or --CH 2  --CH 2  --, to form a ring by binding to the phenyl substituent, at meta with respect to R 1  ; and R 3  is H, alkyl (1-6C) or --OR 4  (if R 2  is different from --OR 4 ); with R 4  being H or alkyl (1-6C); R 5  being H, alkyl (1-6C), --CH 2  COOH, --C 6  H 5  COOH or --C 6  H 5  SO 3  H; and R 6  is H, alkyl (1-6C) or --CH 2  COOH; or n is 1, R 1  is --OR 4 , R 2  is --CH 2  --O--, in order to form a ring by bonding of O with the phenyl substituent, at meta with respect to R 1  ; and R 3  is H or --OR 4 . Application to assaying the SOD activity in a sample, specially a biological sample, particularly by a single measurement and one calibrating curve.

The present invention relates to the assaying of superoxide dismutase(SOD) activity.

It relates more particularly to a new process for the assay of SODactivity, especially in biological samples, using auto-oxidizablecompounds which are defined below, to kits for the implementation ofthis process and to new compounds which can be used in this process, andto their preparation.

The tissues of aerobic organisms, and those of the human organism inparticular, are the site of continual production of superoxide ion O₂.⁻,which results from the process of cell respiration and from numerousessential metabolic pathways.

This production of superoxide ion increases considerably when theseorganisms are subjected to an "oxidizing stress" of toxicological origin(hyperoxia, irradiation, intoxication by xenobiotics which generate freeradicals, in particular) or of physiopathological origin (inflammation,ischaemia, post-ischaemic reperfusion, in particular).

It is accepted by the entire scientific community that the superoxideion is highly toxic, although the mechanisms underlying this toxicityremain the subject of controversy (reference 1).

In order to protect themselves against the harmful effects of thesuperoxide ion, aerobic organisms possess enzymatic systems, thesuperoxide dismutases (SODs), which catalyse the breakdown of thesuperoxide ion in accordance with the following dismutation reaction:

    2O.sub.2..sup.- +2 H.sup.+ →H.sub.2 O.sub.2 +O.sub.2

Because of the major protective role of this dismutation reaction withregard to the toxicity of the superoxide ion, the catalytic activity ofintra- and extra-cellular SODs determines the survival of the tissues ofan aerobic organism (references 2 to 5).

The assay of SOD activity is therefore of prime interest to biologicalresearchers and to laboratories of clinical chemistry, on condition thatit should be possible to carry it out using a method which is sensitive,specific, rapid and relatively simple.

Numerous methods for assaying SOD activity have been published andcommented on (references 6 to 10), but it is generally accepted thatthere does not yet exist a satisfactory compromise between reliabilityand complexity in the existing methods, since the measurement of SODactivity poses two major problems which none of the methods published todate has been able to resolve in a simple fashion.

The first problem is that of the spontaneous decomposition of the solesubstrate, the superoxide ion, which is very rapid at a pH of about 7and which, although highly decelerated, remains significant at a pH of9, the pH above which certain types of natural SODs are rapidlydeactivated. To obtain a stationary concentration which is sufficientlystable therefore requires the presence of a dynamic source of superoxideion in the assay solution.

Electrolytic sources require complex equipment and a prior extraction ofthe SOD catalyst, which is prohibitive in clinical chemistry.

Photochemical sources, such as riboflavin, lead to methods which cannotbe standardized and which are complex to carry out.

Enzymatic sources, such as xanthine oxidase, require solutions to beprepared at the time of use. They are expensive, a source of artifacts,and are difficult to automate.

Purely chemical sources come down to auto-oxidizable compounds such as6-hydroxydopamine, pyrogallol, hydroxylamine or the sulfite ion, whoseautoxidation is generally inhibited by SOD.

These chemical reagents rarely give results which are precise andsensitive on crude biological extracts, and they require theextemporaneous preparation of an anaerobic solution of the reagent;their use is therefore difficult to automate.

The second problem is related to the fact that it is difficult tomeasure directly ("direct" assay) the disappearance of the superoxidesubstrate O₂.⁻ as a function of time, or the appearance of itsdismutation products, hydrogen peroxide H₂ O₂ and oxygen, because of theconcentrations and the pH values which are accessible under theconditions of an assay of SOD activity.

The only "direct" spectrophotometric method is that of MARKLUND(references 11 and 12), which measures the rate of disappearance of thesuperoxide ion at pH 9.5, which does not permit the assay of all naturalSODs.

This method requires a rapid spectrophotometer. The high concentrationsof superoxide ion and the wavelength (250 nm) which are used make themethod relatively imprecise, which obliges the experimenter to carry outa number of measurements in order to obtain an average value. Inaddition, it is necessary to add catalase to avoid the deactivation ofthe SOD-Cu/Zn, and it is necessary to reprepare a new solution ofpotassium superoxide immediately before each assay.

This direct spectrophotometric method is therefore complicated to carryout, and it does not appear possible to improve it in any significantway.

The corollary of this is that the popular methods, in other words thosewhich are used most often, are generally indirect: they rest on thecompetition between the SOD activity which it is attempted to measureand the reaction of the superoxide ion with a chromogenic scavenger suchas ferrocytochrome C or nitro blue tetrazolium (NBT), the reactionproduct of which can be measured by UV/visible spectrophotometry. Achemiluminescent scavenger, such as luminol or lucigenin, may also beused with a greater sensitivity of the measurement, but the problems ofinterferences and of instrumental availability preclude the use of suchmethods by a non-specialist laboratory.

In addition to the often inadequate specificity of the chromogenicreagents with regard to the superoxide ion, the principal disadvantageof the competitive assays is that they require a linearization of theinhibition (by SOD) values, over 5 or 6 samplings of increasingdilutions.

Finally, a certain number of immunoassays of SOD-Cu/Zn or of SOD-Mn areknown.

Some of them are very sensitive, but they give no information on theenzymatic activity of the sample, which depends in particular on thedegree of possible deactivation of the enzyme. Moreover, they take along time to carry out and are relatively complex.

Artifacts of measurements and demultiplication of the assay aretherefore intrinsically linked to all the spectrophotometric methodswhich are accessible to the majority of laboratories, which does notsatisfy the requirements of research laboratories and remainsincompatible with the imperatives of standardization in clinicalchemistry.

If use is made of UV/visible spectrophotometry, it seems difficult--oreven impossible--to develop a method of assay which is at one and thesame time sensitive, more specific, more simple and more reliable thanthe existing methods, unless recourse is had to the monitoring of thekinetics of a reaction which is activated by SOD activity rather than tothat of a reaction which is inhibited by SOD activity or to a "direct"method.

In this respect, a recent method (references 13 and 14), which uses anauto-oxidizable colourant, haematoxylin, differs from others in themeasurement, for which it utilizes activation of the autoxidation of thereagent by SOD activity.

In effect, haematoxylin undergoes autoxidation at a pH>6.5 (reference13). The rate of autoxidation factor of close to 100 between pH 7 and 9.SOD inhibits this autoxidation at a pH<8.1, while activating it at apH>8.1. This observation led to the development of an assay, based onthe spectrophotometric monitoring of the formation of haematein at 560nm, which is accelerated by SOD at alkaline pH. The application of thismethod is complex because of the instability of the reagent, and itremains dependent on a phase of preparation of the sample (dialysis) inorder to remove the interferences which are due to the presence ofreducing compounds such as, for example, glutathione.

Moreover, the low sensitivity of the assay (at a pH>8.1), linked in partto the decomposition of the measured oxidation product, haematein, butalso to the presence of two sites of potential oxidation (catechols),limit the interest and the exploitation of this method.

In conclusion, the haematoxylin method, although having a certaininterest, possesses some invalidating disadvantages which do not allowit to be used in general for assaying SOD activity, in particular inbiological samples.

The objectives of the present invention are, in particular:

to make available a reagent whose autoxidation product is stable duringmeasurement;

to increase the sensitivity of the assay in relation to a method usinghaematoxylin (under conditions in which autoxidation is activated);

to enable the elimination of the most likely possible interferences,such as those linked to the presence of mercaptans such as glutathione,for example;

to develop a process which uses a reaction medium with a pH of less thanor equal to 9, so as to permit an assay of the activity of known SODswithout the risk of rapid deactivation.

These objectives are attained in accordance with the invention, whichutilizes the chemical reagents defined below, which have the property ofundergoing autoxidation in an oxygenated solution, to autoxidation in anoxygenated solution, to lead to a chromophoric product, the kinetics ofwhose appearance is increased in the presence of a catalyst of the SODtype.

The structure of these reagents is characterized, in particular, by anaromatic ring with a catechol function, which is linked via a tertiarybenzyl carbon to an aromatic ring which is substituted with anelectron-donating group.

The autoxidation product of these reagents has an absorption wavelengthwhich is very different from that characterizing the reagent itself.

The invention therefore provides a process which employs these reagents,which enables the SOD activity of any catalyst to be assayed in aqueousmedium, using a single UV/visible absorption measurement, which processis markedly more simple in its application than that of the otherspectrophotometric methods which have been described beforehand.

The invention also provides a process for the stabilization of a stocksolution of the chemical reagents defined below, by means of boronderivatives, in particular boric acid and its derivatives such asphenyl-boric acid.

The invention furthermore provides a process for assaying the SODactivity of the type described above, in the presence of mercaptans, byvirtue of the use of a rapid reagent for thiol groups, which enables theelimination of the corresponding interferences, in particular thosewhich are caused by the presence of glutathione or albumin in thesample.

According to one of its aspects, the subject of the invention is aprocess for the assay of superoxide dismutase (SOD) activity in a liquidmedium, which process utilizes activation of the autoxidation of areagent by SOD activity, characterized in that the said reagent is acompound conforming to the general formula (I): ##STR1## in which:either:

n=1 or 2,

R¹ =--OR⁴ or --NR⁵ R⁶,

R² =hydrogen, --OR⁴ alkyl (with 1 to 6 carbon atoms) or else --CH₂ -- or--CH₂ --CH₂ --, to form a ring by linking with the phenyl substituent,in the meta position in relation to R¹, and

R³ =hydrogen, alkyl (with 1 to 6 carbon atoms) or --OR⁴ (with theproviso that R² is different from --OR⁴), where

R⁴ =hydrogen or alkyl (with 1 to 6 carbon atoms),

R⁵ =hydrogen, alkyl (with 1 to 6 carbon atoms), --CH₂ COOH, --C₆ H₅ COOHor --C₆ H₅ SO₃ H, and

R⁶ =hydrogen, alkyl (with 1 to 6 carbon atoms) or --CH₂ COOH;

or:

n=1,

R¹ =--OR⁴ (R⁴ being defined as above),

R² =--CH₂ --O--, to form a ring by linkage of the oxygen atom with thephenyl substituent, in the meta position in relation to R¹, and

R³ =hydrogen or --OR⁴ (R⁴ being defined as above).

The compound of general formula (I) above in which n=1, R¹ =--OH, R²=--CH₂ --O-- and R³ =--OH is brazilin which is a commercial product.

The other compounds conforming to the general formula (I) defined abovemay be prepared according to the general procedures which are describedhereinafter, generally from commercial products.

However, the compounds in which R⁵ represents --C₆ H₅ COOH or --C₆ H₅SO₃ H are obtained from the synthon of the para-bromophenylaminophenyltype, substituted in an appropriate manner. This synthon is preparedaccording to the following general procedure.

para-Nitrophenol is alkylated by means of an ester of chloroacetic acid.The derivative obtained is hydrolyzed and then amidated withpara-bromoaniline. The corresponding amide is subjected to the reactionof Smiles (reference 15) to give the corresponding diphenylaminederivative. The latter is reduced, diazotized, and substituted in anappropriate manner to give the desired synthon.

General procedure for the synthesis of compounds of series I (n=1 or 2,R¹ =--OR⁴ or NR⁵ R⁶, R² =hydrogen or alkyl and R³ =hydrogen or alkyl):

5,6-Dihydroxindan-1-one or 6,7-dihydroxy-1-tetralone (prepared fromveratrole), suitably protected by an alkyl or aralkyl group such as, forexample, the methyl or benzyl group, or by a group of silyl type suchas, for example, the tert-butyldimethylsilyl group, is treated, eitherdirectly or after prior mono- or bis-alkylation (of the α position ofthe ketone function) using an alkylating agent such as methyl iodide,for example, by the organomagnesian reagent corresponding to phenylbromide substituted in the para position by an oxygen-containing groupsuch as, for example, the methoxy group, or a nitrogen-containing groupsuch as, for example, the dimethylamino group.

The intermediates thus obtained are reduced and then deprotected to givethe compounds of series I.

The following reaction scheme illustrates this procedure. ##STR2## i: R¹PhMgBr (R¹ in para position) ii: reduction then deprotection

iii: base, R² X

iiii: base, R³ X

P: protecting group

General procedure for the synthesis of compounds of series II (n=1 or 2,R¹ =--OR⁴ or --NR⁵ R⁶, R² =hydrogen or alkyl and R³ =--OR⁴).

These compounds are obtained by hydration of the alkenes which areintermediates in the synthesis of compounds of series I, afterhydroboration and oxidation; the alcohols thus obtained are then, ifappropriate, alkylated with an alkyl halide such as, for example, methyliodide and then deprotected.

The following reaction scheme illustrates this procedure. ##STR3## i: 1)BH₃ : 2) H₂ O₂, NaOH ii: deprotection

iii: base, R⁴ X

P: protecting group

General procedure for the synthesis of compounds of series III (n=1 or2, R¹ =--OR⁴ or --NR⁵ R⁶ and R² =--CH₂ -- or --CH₂ --CH₂ -- forming aring by linkage with the phenyl substituent, in the meta position inrelation to R¹):

To synthesize these compounds, the product of crotonization between3,4-dihydroxybenzaldehyde, protected in a suitable fashion, and1-tetralone or 1-indanone which are suitably substituted, is reduced,either directly or indirectly (after preparation of the superiorhomologue by way of a cyclopropane derivative), by catalytichydrogenation to give the corresponding saturated derivative. The latteris cyclized in the presence of polyphosphoric acid and the alkene thusobtained is reduced and then deprotected to give the desired compound ofseries III.

The following reaction scheme illustrates this procedure. ##STR4## i:H^(+ii:) reduction (or preparation of the superior homologue thenreduction)

iii: polyphosphoric acid

iiii: reduction

iiiii: deprotection

P: protecting group

General procedure for the synthesis of compounds of series IV (n=1, R¹=--OH, R² =--CH₂ --O--, to form a ring by linkage of the oxygen atomwith the phenyl substituent, in the meta position in relation to R¹ andR³ =hydrogen or --OR⁴):

These compounds can be synthesized from brazilin according to thefollowing reaction scheme: ##STR5##

In this scheme:

Bn represents a protecting group of the phenolic OH groups, such as thebenzyl group,

R⁴ X' represents an alkylating agent.

After having protected the 3 phenolic hydroxyls in the form, forexample, of benzyl ether, the tertiary alcohol group is eliminated inthe form of phenyl thiono carbonate, for example, or else alkylatedusing, for example, methyl iodide. The desired product is obtained,after deprotection, by catalytic hydrogenation in the presence, inparticular, of palladium on carbon, which is concomitant with areduction in the case of brazilane.

Examples 9 and 10 of the experimental section which follows illustratethe preparation of compounds of this type.

General procedure for the synthesis of compounds of series V (n=1, R¹=--O--alkyl, R² =--CH₂ --O--, to form a cycle by linkage of the oxygenatom with the phenyl substituent, in the meta position in relation to R¹and R³ =hydrogen or --OR⁴):

Brazilin or one of its derivatives in which R¹ =OH and R³ is differentfrom OH is protected on its two catecholic hydroxyls by a methylenebridge, for example by means of formaldehyde. The derivative obtained isalkylated on its unprotected hydroxyl by means of an alkylating agentsuch as, for example, an alkyl halide and then deprotected in acidmedium.

Example 11 of the experimental section which follows illustrates thepreparation of a compound of this type, in which R¹ =--O-methyl and R³=--OR⁴ where R⁴ =hydrogen.

All of the brazilin derivatives of series IV and V are obtained bychemical modification of its hydroxyl groups. The modifications whichare carried out make it possible to modulate the sensitivity of theassay of SOD activity.

When the assay of SOD activity has to be carried out in a mediumcontaining one or more compounds which comprise one or more thiolgroups, such as, in particular, a biological medium, the reducing powerof the latter groups is capable of causing interferences which it isappropriate to remove.

Such interferences may easily be avoided by adding, to the medium inwhich the assay is carried out, at least one reagent chosen fromcompounds of the pyridinium and quinolinium types, whose use as"mercaptan scavengers" is the subject of the French Patent Applicationfiled on 29 Nov. 1991 in the name of BIOXYTECH under the No. 9114782,and whose title is: "Mercaptan-scavenging reagents, their preparationand their applications".

These reagents correspond to the following criteria:

specificity for mercaptans under the operating conditions used,

rapidity of reaction,

non-interference with the other reagents used in the assay of SODactivity.

More precisely, according to another of its aspects, the inventionrelates to a process for the assay of superoxide dismutase (SOD)activity in a liquid medium containing one or more mercaptan(s),especially a biological medium, which process utilizes activation of theautoxidation of a reagent by SOD activity, characterized in that thesaid reagent is a compound conforming to the general formula I definedabove and in that the medium additionally contains a quantity, which iscapable of totally scavenging the said mercaptans by S-alkylation, of atleast one compound conforming to the general formula II: ##STR6## inwhich: Z¹ =alkyl (with 1 to 6 carbon atoms), benzyl, p-nitrobenzyl,phenyl, o,p-dinitrophenyl, --CH₂ --COOH or --CH₂ --CH₂ --COOH;

Z⁴ =hydrogen and

Z⁵ =hydrogen or alkyl (with 1 to 6 carbon atoms), or

Z⁴ and Z⁵ form, together with the two intermediate carbon atoms, aphenyl ring;

X=halide, sulphonate, fluorosulphonate, phosphonate, tetrafluoroborateor tosylate; and

either

Z² =vinyl, and

Z³ =hydrogen or alkyl (with 1 to 6 carbon atoms) (2-vinyl compounds),

or

Z³ =vinyl, and

Z² =hydrogen or alkyl (with 1 to 6 carbon atoms) (4-vinyl compounds).

A sulphonate is understood to be an anion of general formula R--SO₃ ⁻ inwhich R represents, for example, trifluoromethyl.

Some of these compounds are known, or they can be synthesized by analogywith processes used for the synthesis of known compounds.

The 2-vinyl compounds may thus be synthesized as follows.

The initial derivative (substituted quinoline or pyridine) is subjectedto the method of Comins (reference 16), using vinylmagnesium bromide.The compound thus obtained is oxidized with, for example,tetrachloro-1,4-benzoquinone (parachloranil) or sulphur S₈. Thealkylation of the heterocyclic nitrogen is carried out using analkylating agent, for example trimethyloxonium tetrafluoroborate, methylsulfate, benzyl bromide, fluorodinitrobenzene, sodium iodoacetate orsodium 3-bromopyruvate, which are chosen according to the meaning of Z¹.

Example 12 of the experimental section which follows illustrates thepreparation of a compound of this type.

Moreover, the 4-vinyl compounds can be synthesized as follows.

The corresponding heterocyclic aldehyde (aldehyde function, --CHO, inposition 4) is subjected to the Wittig reaction with, for example,triphenylmethylphosphonium bromide, or to the Peterson reaction with,for example, chloromethyltrimethylsilane. The alkylation of theheterocyclic nitrogen is carried out using an alkylating agent, forexample trimethyloxonium tetrafluoroborate, methyl sulphate, benzylbromide, fluorodinitrobenzene, sodium iodoacetate or sodium4-bromopyruvate, which are chosen according to the meaning of Z¹.

Example 13 of the experimental section which follows illustrates thepreparation of a compound of this type.

The process according to the invention, especially when it employs atleast one "mercaptan scavenger" as defined above, makes it possible toassay the SOD activity of any catalyst in aqueous medium, using a singlemeasurement of the UV/visible absorption, which is markedly more simpleto carry out than the spectrophotometric methods described beforehand.

This new assay process thus constitutes a tool of choice for biologicalresearch in general and in clinical chemistry, in particular for thedevelopment of assay kits.

In the latter case, this assay is capable of providing useful medicalinformation in physiopathological conditions such as diseases ofinflammatory or ischemic origin, states of shock, infectious states,systemic diseases and autoimmune diseases, tumours, states ofmalnutrition, diseases associated with aging, degenerative disorders,haemolytic anaemias and the syndromes of intoxication by environmentalpollutants or by medicaments.

The human samples on which this method can be utilized are, inparticular, erythrocytes, blood plasma, platelets, leucocytes, synovialfluid, cerebrospinal fluid, urine or any tissue extract.

Moreover, this assay method can be used to carry out the pharmacokineticstudies which are necessary for the development and use of newmedicaments which have SOD activity.

This assay method can also be used for the quality control of cosmeticor pharmaceutical preparations which have SOD activity.

In a general manner, this assay method can be used by researchers on anybiological sample or any artificial solution which is liable to have SODactivity.

According to a preferred embodiment, the process for the assay of thesuperoxide dismutase activity of a sample, especially a biologicalsample, resides in a spectrophotometric measurement of the rate ofautoxidation of a compound of general formula I above, in the absenceand in the presence of the said sample.

More precisely, according to a preferred embodiment, the inventionrelates to a process for the assay of superoxide dismutase (SOD)activity in a liquid sample, characterized in that it essentiallycomprises the steps consisting in:

1) determining the maximum rate of autoxidation Vs of a compound ofgeneral formula I in the presence of the sample, by means of the changein the absorbance as a function of time, at the wavelength whichcharacterizes the appearance of the autoxidation product of the compoundof general formula I used, in a reaction medium which is buffered to apH value of from 8.0 to 9.0, triggering the reaction by the addition ofan aliquot of a stock solution of the compound of general formula I;

2) determining, under the same conditions, the maximum autoxidation rateV_(c) of the same compound in the absence of the sample; and

3) determining the SOD activity of the sample by means of the ratesV_(s) and V_(c) obtained and of a calibration curve established underthe same operating conditions.

The reaction medium consists advantageously of an amine buffer such as,for example, 2-amino-2-methyl-1,3-propanediol (AMPD) containingdiethylenetriaminepentaacetic acid (DTPA), 0.1 mM, and allowing the pHto be fixed at a value in the range from 8.0 to 9.0 at the measurementtemperature, for example by adding sodium hydroxide solution orhydrochloric acid according to the nature of the buffer underconsideration.

This reaction medium must be equilibrated at the working temperature andsaturated with air at this temperature.

The working temperature chosen, for example 37° C., must be maintainedconstant during the measurements.

The reaction is triggered by the addition to the reaction medium (withor without SOD) of an aliquot of a stock solution of one of thecompounds of general formula I in an aqueous solution or in awater-miscible organic solvent such as, for example, acetonitrile,dimethylformamide or dimethyl sulfoxide, or else in a mixture of such anorganic solvent with distilled water.

In order to avoid an extemporaneous preparation of this stock solutionor an anaerobic preparation of the latter, the stock solution can beprepared in the presence of a boron derivative such as, for example,boric acid which enables a reagent solution to be obtained which isstable for a period which depends on the respective concentrations ofthe reagent and the boron derivative, on the respective nature of thereagent and of this derivative and on the storage conditions of thestock reagent solution. Thus a solution of the reagent BXT01050described below (Example 7), prepared in a 50:50 (volume/volume) mixtureof dimethyl sulfoxide/distilled water and containing boric acid, suchthat the ratio of the concentrations of boric acid and of reagent areequal to 150, is stable for at least one month if the solution is storedat between 0° and 8° C.

After homogenization of the solution, the change in the absorbance isrecorded as a function of time at the wavelength which characterizes theappearance of the autoxidation product of the reagent used (for example525 nm for the reagent BXT01050 and 539 nm for brazilin), in order todetermine the maximum rate of autoxidation.

The determination of SOD activity in a biological sample can be carriedout by determining the ratio or the difference of the rates measured inthe presence (V_(s)) and in the absence (V_(c)) of the sample.

This ratio or this difference can be recorded, for example, on theordinate of a calibration curve which has been established under thesame operating conditions using, for example, a SOD standard.

This calibration curve can be curveted by representing, as a function ofthe concentration of the standard used, the variation in the ratio or inthe difference of the autoxidation rates measured in the presence(V_(s)) and in the absence (V_(c)) of this standard.

The ratio V_(s) /V_(c) varies with the concentration of SOD according toa hyperbolic function of the type y=1+x/(ax+b), in which x representsthe concentration of SOD and a and b are constants. The inverse of thedifference in the rates as a function of the inverse of theconcentration of SOD is one of the possible linearizations of thisfunction.

According to the intensity of the SOD activity to be measured, thesensitivity of the assay in the pH range used (8.0 to 9.0) can beadapted by modifying, for example, one or more of the followingparameters: concentration of the compound of general formula I chosen,concentration of the buffer and measurement temperature.

The rate of autoxidation of the reagent used may be modulated also byvarying the concentration of boron derivative in the reaction medium.

The existence in a sample, especially a biological sample, of compoundswhich interfere with the assay is most often evident in a decrease inthe rate measured in the presence of the sample in relation to thatmeasured in its absence.

In the case of interferences which are linked to the presence ofmercaptans such as, for example, glutathione, the introduction into thereaction medium of a "mercaptan-scavenging reagent" as defined above isthen necessary.

Thus 1,4,6-trimethyl-2-vinylpyridinium tetrafluoroborate (compound ofExample 12), for example, will be incorporated in a concentration whichis fifty times greater than that of glutathione for the assay of SODactivity in an erythrocyte lysate.

If the existence is suspected in a sample, especially a biologicalsample, of a compound which is liable to introduce an artifactualactivation of the autoxidation of the compound of general formula I, orchromogenic compound, a combination such as that described below may beundertaken, which involves making a second measurement carried out underconditions in which the autoxidation is inhibited.

Likewise, in the case of the examination of media, especially biologicalmedia, which have a low SOD activity, the sensitivity and the precisionof the assay can be increased by combining, with the measure made underconditions of activation of the autoxidation, a measurement carried outunder conditions in which the autoxidation is inhibited.

Therefore the invention likewise relates to a process of the typedescribed above, characterized in that it also comprises a measurementcarried out under conditions in which the autoxidation is inhibited.

The reaction medium used for this second measurement differs from themedium described above by the use of a buffer at a pH within the rangefrom 7.2 to 7.8, especially a phosphate buffer consisting, for example,of a suitable mixture of potassium dihydrogen phosphate and dipotassiumhydrogen phosphate, all other things being equal.

Under these operating conditions the maximum autoxidation rate of thecompound of general formula I used is measured in the absence (V'_(c))and in the presence (V'_(s)) of the sample. The determination of the SODactivity in the sample is then obtained by means of (V_(s) -V_(c)-V'_(s) +V'_(c)).

As previously, the value obtained can be recorded on the ordinate of acalibration curve which has been established under the same experimentalconditions and which gives the variation of (V_(s) -V_(c) -V'_(s)+V'_(c)) as a function of the concentration of the standard used.

The new assay method according to the invention which uses as reagent acompound of general formula I is, on the basis of its principle, amethod for specific measurement of SOD activity. It is sensitive, rapidand easy to carry out, and it is, moreover, entirely capable ofautomation; it is therefore suitable for the treatment of large seriesof samples as may be the case in clinical chemistry.

Moreover, the interferences which are linked to the presence ofmercaptans and are frequently encountered in biological samples caneasily be removed by the use of at least one "mercaptan-scavengingreagent" as defined above, without another supplementary phase oftreatment of the sample.

Finally, the operating conditions of this assay process, which are ofhigh modulability, impart to the process a flexibility in use whichenables it to be adapted to very diverse samples, especially biologicalsamples, and therefore to take account of the particular possiblerequirements of certain users, as is the case in research.

Application examples of the new process for the assay of SOD activity,using seven compounds of general formula I, in the case of anerythrocyte lysate, are given in an illustrative capacity in theexperimental section which follows.

The invention also relates to a kit for the implementation of theprocess for assaying SOD activity according to the invention.

This kit essentially comprises, as reagent, a compound of generalformula I as defined above.

According to an advantageous embodiment, the kit for the implementationof the process for assaying SOD activity according to the inventionadditionally comprises one or more mercaptan-scavenging compound(s) ofgeneral formula II as defined above.

According to a preferred embodiment, the invention also relates to a kitfor the implementation of the process for assaying SOD activityaccording to the invention, characterized in that it essentiallycomprises:

a compound of general formula I in acidic solution or in a powder,

one or more mercaptan-scavenging compound(s) of general formula II, insolution or as a powder, and

a buffer based on AMPD, which buffers in the pH range from 8.0 to 9.0.

The invention also relates to the compounds of general formula I asdefined above, with the proviso that, when n=1 and R² =CH₂ --O--, R¹ andR³ may not simultaneously represent --OR⁴ where R⁴ =hydrogen.

The attached figures represent calibration curves for the implementationof the process according to the invention, using six different compoundsof general formula I, according to the mode of operation which isdescribed, respectively, in Examples 14 to 19 and 21 of the experimentalsection which follows.

These calibration curves were obtained according to two differentmethods which are given here by way of example, namely:

First method:

curveting the inverse of the "SOD concentration", expressed in ml/U(U=unit of SOD activity) on the abscissa and

curveting the inverse of V_(s) -V_(c), V_(s) and V_(c) being as definedabove, expressed in min/ΔAbs. (ΔAbs.=change in absorbance) on theordinate.

Second method:

curveting the "SOD concentration", expressed in U/ml (U=unit of SODactivity), on the abscissa and

curveting the ratio V_(s) /V_(c), V_(s) and V_(c) being as definedabove, on the ordinate.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 represents the calibration curve of the compound BXT01041,obtained according to the first method;

FIG. 2 represents the calibration curve of the compound BXT01048,obtained according to the first method;

FIG. 3 represents the calibration curve of the compound BXT01049,obtained according to the first method;

FIG. 4 represents the calibration curve of the compound BXT01050,obtained according to the first method;

FIG. 5 represents the calibration curve of brazilin, obtained accordingto the first method;

FIG. 6 represents the calibration curve of brazilane, obtained accordingto the first method; and

FIG. 7 represents the calibration curve of the compound BXT01050,obtained according to the second method.

EXPERIMENTAL SECTION I. Preparation of Compounds of General Formula I

All of the reactions were carried out under an inert nitrogen atmosphereunless otherwise indicated.

The mass spectra (MS) were recorded on an R10-10B apparatus from thecompany Nermag. The ionization mode used is either electron impact (EI)at 70 electron-volts or chemical ionization (CI), unless otherwiseindicated.

The ¹ H NMR spectra were recorded on a Gemini-200 type apparatus fromthe company VARIAN. The chemical shifts are expressed in ppm in relationto tetramethylsilane. The multiplicities are expressed as follows: "s"for singlet; "d" for doublet; "t" for triplet; and "m" for multiplet.

The melting points (m.p.) were recorded with an apparatus from thecompany Gallenkamp and are given in uncorrected form. Therecrystallization solvents are indicated in brackets.

A. Compounds of Series I EXAMPLE 1 Preparation of5,6-dihydroxy-1-(4-hydroxyphenyl)indane (BXT01041)

5,6-Dimethoxy-3-(4-methoxyphenyl)indene

A small portion of 4-bromoanisol is added to a suspension of magnesium(0.24 g, 10 mmol) in 5 ml of anhydrous THF. the mixture is heatedslightly to initiate the reaction, then the rest of the brominederivative (1.87 g, 10 mmol) dissolved in 5 ml of THF is added dropwiseover the course of 15 min. After refluxing for 1 h, the magnesium hasdisappeared. The mixture thus obtained is cooled to room temperature,and a solution of 5,6-dimethoxyindan-1-one (1.92 g, 10 mmol) in 10 ml ofanhydrous THF is added. The brown solution obtained is stirred for 20 hunder these conditions and then hydrolysed by addition of saturated NH₄Cl solution. The mixture is extracted with AcOEt. The organic phasescollected are washed with saturated aqueous NaCl solution and dried overNa₂ SO₄. After evaporation of the solvent, a yellow oil (3.43 g) isobtained which is taken up in 5 ml of glacial acetic acid. The solutionis stirred for 15 min at room temperature and then heated at 70° C. for15 min. The solution is cooled to 0° C. and poured slowly into 50 ml ofsaturated NaHCO₃ solution. The mixture is extracted with AcOEt. Theorganic phases collected are washed with saturated aqueous NaHCO₃solution, dried over MgSO₄ and evaporated. The crude product (2.84 g)thus obtained is purified by chromatography, eluting with a 1:3 mixtureof AcOEt/hexane to give a white solid (1.97 g, 70%).

Physical characteristics:

*m.p.: 111.0°-112.0° C. (AcOEt/hexane=1:3). ¹ H NMR (CDCl₃): 3.62 (d,J=2.16 Hz, 2H); 4.05 (s, 3H); 4.08 (s, 3H); 4.12 (s, 3H); 6.58 (t,J=2.16 Hz, 1H); 7.19 (d, J=8.67 Hz, 2H); 7.29 (s, 1H); 7.32 (s, 1H);7.72 (d, J=8.67 Hz, 2H). *MS (EI): 282 (100), 267 (55), 251 (19), 181(24), 165 (35), 152 (51), 84 (53).

5,6-Dimethoxy-1-(4-methoxyphenyl)indane

A solution of 5,6-dimethoxy-3-(4-methoxyphenyl)indene (1.5 g, 5.32 mmol)in 60 ml of ethanol is hydrogenated in an autoclave in the presence ofpalladium on carbon (10%, 25 mg) under 5 bars of hydrogen for 1.5 h.After filtration of the catalyst, the evaporation of the solvent givesan oil which gradually solidifies (1.5 g, 100%).

Physical characteristics:

*¹ H NMR (CDCl₃): 1.99 (m, 1H); 2.45 (m, 1H); 2.90 (m, 2H); 3.72 (s,3H); 3.79 (s, 3H); 3.87 (s, 3H); 4.24 (t, J=7.97 Hz, 1H); 6.48 (s, 1H);6.82 (s, 1H); 6.84 (d, J=8.70 Hz, 2H); 7.50 (d, J=8.70 Hz, 2H). *MS(EI): 284 (100), 269 (26), 253 (59), 241 (17), 177 (21), 165 (35), 152(28), 155 (28), 84 (25), 77 (26).

5,6-Dihydroxy-1-(4-hydroxyphenyl)indane

A solution of BBr₃ in CH₂ Cl₂ (1.0M, 3.1 ml, 3.1 mmol) is added dropwiseto a solution of the phenylindane obtained above (500 mg, 0.86 mmol) in2 ml of CH₂ Cl₂, cooled to -70° C. The red solution obtained is heatedto room temperature and stirred for 0.5 h at this temperature. Thereaction mixture is poured into a mixture of ice-water and AcOEt. Theaqueous phase is saturated with NaCl and then extracted twice withAcOEt. The organic phases collected are washed with saturated aqueousNaCl solution, dried over MgSO₄ and evaporated to dryness to give ayellow oil (480 mg) which is chromatographed (eluent: AcOEt/hexane=1:1)to give the expected compound (white solid, 400 mg, 93%).

Physical characteristics:

*¹ H NMR (acetone-d6): 1.90 (m, 1H); 2.41 (m, 1H); 2.79 (m, 2H); 4.08(t, J=7.97 Hz, 1H); 6.39 (s, 1H); 6.75 (s, 1H); 6.77 (d, J=8.70 Hz, 2H);6.98 (d, J=8.70 Hz, 2H); 7.77 (s, broad, 3H). *MS (EI): 242 (100), 225(61).

EXAMPLE 2 Preparation of5,6-dihydroxy-1-(4-hydroxyphenyl)-2,2-dimethylindane (BXT01045)

5,6-Dimethoxy-2-methylindan-1-one and5,6-dimethoxy-2,2-dimethylindan-1-one

A mixture of lithium diisopropylamide (LDA, 1.5M in cyclohexane, 7.5 ml,11.3 mmol) and 15 ml of anhydrous THF is cooled to -78° C. A solution of5,6-dimethoxyindan-1-one (1.92 g, 10 mmol) in 5 ml of THF is addeddropwise thereto. The mixture is stirred for 30 min at -78° C. Methyliodide (0.93 ml, 15 mmol) is added. After 30 min at -78° C. the solutionis heated to room temperature and stirred at this temperature for 4 h.The solution is again cooled to -78° C. and the same quantity of LDA asbefore is added dropwise. After 30 min, MeI (0.93 ml, 15 mmol) is added.The mixture is stirred for 30 min at -78° C. and then for 4 h at roomtemperature. The reaction mixture is hydrolysed by the addition ofsaturated aqueous NH₄ Cl solution. It is extracted with AcOEt. Theorganic phases are washed with saturated aqueous NaCl solution, driedover MgSO₄ and evaporated to dryness to give a yellow solid (2.23 g).Chromatography, using a 1:9 mixture of AcOEt/hexane as eluent, gives5,6-dimethoxy-2,2-dimethylindan-1-one (1.35 g, 61%) and5,6-dimethoxy-2-methylindan-1-one (0.43 g, 21%).

Physical characteristics of 5,6-dimethoxy-2,2-dimethylindan-1-one:

*¹ H NMR (CDCl₃): 1.64 (s, 6H); 2.85 (s, 2H); 3.85 (s, 3H); 3.91 (s,3H); 6.80 (s, 1H); 7.12 (s, 1H). *m.p.: 134.0°-134.5° C. (EtOH).

Physical characteristics of 5,6-dimethoxy-2-methylindan-1-one:

*¹ H NMR (CDCl₃): 1.22 (d, J=7.41 Hz, 3H); 2.63 (m, 2H); 3.23 (dd,J=9.24-16.85 Hz, 1H); 3.84 (s, 3H); 3.90 (s, 3H); 6.81 (s, 1H); 7.11 (s,1H). *m.p.: 106.0°-106.5° C. (EtOH).

1-Hydroxy-5,6-dimethoxy-1-(4-methoxyphenyl)-2,2-dimethylindane

A small portion of 4-bromoanisole is added to a suspension of magnesium(72 mg, 3 mmol) in 6 ml of anhydrous THF. The mixture is heated atgentle reflux to initiate the reaction, then the remainder of thebrominated derivative (0.56 g, 3 mmol), in solution in 5 ml of THF, isadded dropwise over the course of 15 min. After reflux for 1 h, themagnesium has been consumed. 5,6-Dimethoxy-2,2-dimethylindan-1-one (0.66g, 3 mmol) is added in small portions. The solution is stirred for 1 hat room temperature, then heated at reflux for 4 h. 10 ml of saturatedaqueous NH₄ Cl solution are added at 0° C. The mixture is extracted withAcOEt. The organic phases collected are washed with saturated aqueousNaCl solution, dried over MgSO₄ and evaporated to give a yellow oil(1.20 g). Purification by chromatography (eluent: AcOEt/hexane=1:5)enables the isolation of the expected product (0.65 g, 66%).

Physical characteristics:

*¹ H NMR (CDCl₃): 0.62 (s, 3H); 1.13 (s, 3H); 2.60 (d, J=15.10 Hz, 1H);2.86 (d, J=15.10 Hz, 1H); 3.77 (s, 3H); 3.79 (s, 3H); 3.89 (s, 3H); 3.91(s, 1H); 6.71 (s, 1H); 6.79 (s, 1H); 6.83 (d, J=9.00 Hz, 2H); 7.19 (d,J=9.00 Hz, 2H).

5,6-Dimethoxy-1-(4-methoxyphenyl)-2,2-dimethylindane

Sodium borohydride (1.40 g, 36.7 mmol) and aluminum trichloride (0.82 g,6.1 mmol) are added to a solution of the preceding product (0.39 g, 1.25mmol) in 15 ml of anhydrous THF. The mixture is heated at reflux. Itimmediately turns pink, then red and finally becomes colorless. After 20h of reflux, the reaction mixture is poured into a mixture of 20 g ofice and 5 ml of HCl (1N). The aqueous phase is saturated with NaCl andextracted with AcOEt. The organic phases are washed with saturatedaqueous NaCl solution, dried over MgSO₄ and evaporated to dryness togive a colorless oil. Chromatography, eluting with a 1:10 mixture ofAcOEt-hexane, gives the expected product (0.39 g, 66%).

Physical characteristics:

*¹ H NMR (CDCl₃): 0.63 (s, 3H); 1.19 (s, 3H); 2.72 (s, 2H); 3.74 (s,3H); 3.79 (s, 3H); 3.87 (s, 3H); 3.91 (s, 1H); 6.56 (s, 1H); 6.78 (s,1H); 6.83 (d, J=8.86 Hz, 2H); 6.98 (d, J=8.86 Hz, 2H).

5,6-Dihydroxy-1-(4-hydroxyphenyl)-2,2-dimethylindane

A solution of BBr₃ in CH₂ Cl₂ (1.0M, 4.86 ml, 4.86 mmol) is addeddropwise to a solution of the derivative obtained above (0.39 g, 1.25mmol) in 2 ml of CH₂ Cl₂, cooled to -70° C. The red solution obtained isheated to room temperature and stirred for 1 h at this temperature. Thereaction mixture is poured into a mixture of ice-water and AcOEt. Theaqueous phase is saturated with NaCl and then extracted twice withAcOEt. The organic phases collected are washed with saturated aqueousNaCl solution, dried over MgSO₄ and evaporated to dryness to give aslightly red oil (410 mg) which is chromatographed (eluent:AcOEt/hexane=1:1) to give the expected compound (0.29 g, 86%).

Physical characteristics:

*¹ H NMR (acetone-d6): 0.71 (s, 3H); 1.17 (s, 3H); 2.60 (s, 2H); 3.80(s, 1H); 6.46 (s, 1H); 6.72 (s, 1H); 6.75 (d, J=8.74 Hz, 2H); 6.89 (d,J=8.74 Hz, 2H). *MS (EI): 270 (87), 227 (100), 107 (16).

EXAMPLE 3 Preparation of5,6-dihydroxy-1-(4-hydroxyphenyl)-2-methylindane (BXT01048)

5,6-Dimethoxy-3-(4-methoxyphenyl)-2-methylindene

A small portion of 4-bromoanisole is added to a suspension of magnesium(42 mg, 1.750 mmol) in 5 ml of anhydrous THF. The mixture is heated atgentle reflux to initiate the reaction, then the remainder of thebrominated derivative (330 mg, 1.75 mmol), in solution in 5 ml of THF,is added dropwise over the course of 15 min. After 1 h of reflux,magnesium has been consumed. The mixture thus obtained is cooled to roomtemperature and then a solution of 5,6-dimethoxy-2-methylindan-1-one(300 mg, 1.46 mmol) in 5 ml of THF is added. The brown solution obtainedis stirred for 18 h, then hydrolyzed by the addition of saturatedaqueous NH₄ Cl solution. The mixture is extracted with AcOEt. Theorganic phases collected are washed with saturated aqueous NaClsolution, then dried over Na₂ SO₄. After evaporation of the solvent, ayellow oil (0.54 g) is obtained which is taken up in 2 ml of glacialacetic acid. The solution obtained is stirred for 1 h at roomtemperature. The solution, cooled to 0° C. is poured slowly into 50 mlof saturated NaHCO₃ solution. The mixture is extracted with AcOEt. Theorganic phase is washed with saturated aqueous NaHCO₃ solution, driedover MgSO₄ and evaporated. The crude product (530 mg) thus obtained ispurified by chromatography, eluting with a 1:9 mixture of AcOEt/hexane,to give a colorless oil (0.25 g, 58%).

Physical characteristics:

*¹ H NMR (CDCl₃): 2.09 (s, 3H); 3.35 (s, 2H); 3.82 (s, 3H); 3.86 (s,3H); 3.89 (s, 3H); 6.78 (s, 1H); 7.00 (d, J=8.70 Hz, 2H); 7.04 (s, 1H);7.33 (d, J=8.70 Hz, 2H).

5,6-Dimethoxy-1-(4-methoxyphenyl)-2-methylindane

A solution of the above phenylindene (240 mg, 0.81 mmol) in 5 ml ofAcOEt is hydrogenated under approximately 10⁶ Pa (10 atm) of hydrogen inthe presence of 20 mg of palladium on carbon (10%) for 2 h. The catalystis removed by filtration, then the filtrate is evaporated to dryness togive the pure product expected (0.24 g, 99%).

Physical characteristics:

*¹ H NMR (CDCl₃): 0.69 (d, J=6.96 Hz, 3H); 2.58 (dd, J=6.96-14.30 Hz,1H); 2.80 (m, 1H); 2.96 (dd, J=7.04-14.30 Hz, 1H); 3.75 (s, 3H); 3.77(s, 3H); 3.88 (s, 3H); 4.25 (d, J=7.68 Hz, 1H); 6.63 (s, 1H); 6.80 (d,J=8.78 Hz, 2H); 6.81 (s, 1H); 6.88 (d, J=8.78 Hz, 2H). *MS (EI): 298(100), 283 (25), 267 (44), 252 (7), 238 (12), 167 (42), 149 (42), 135(33), 84 (62).

5,6-Dihydroxy-1-(4-hydroxyphenyl)-2-methylindane

A solution of BBr₃ in CH₂ Cl₂ (1.0M, 3.0 ml, 3.0 mmol) is added dropwiseto a solution of the phenylindane obtained above (0.23 g, 0.77 mmol) in2 ml of CH₂ Cl₂, cooled to -70° C. The red solution obtained is heatedto room temperature and stirred for 0.5 h at this temperature. Thereaction mixture is poured into a mixture of ice-water and AcOEt. Theaqueous phase is saturated with NaCl, then extracted twice with AcOEt.The organic phases collected are washed with saturated aqueous NaClsolution, dried over MgSO₄ and evaporated to dryness to give a yellowoil (0.20 g) which is chromatographed (eluent: AcOEt/hexane=1:1) to givethe expected compound (0.18 g, 91%).

Physical characteristics:

*¹ H NMR (acetone-d6): 0.63 (d, J=6.96 Hz, 3H); 2.45 (dd, J=7.32-14.22Hz, 1H); 2.68 (m, 1H); 2.84 (dd, J=7.04-14.22 Hz, 1H); 4.13 (d, J=7.42Hz, 1H); 6.50 (s, 1H); 6.73 (m, 5H); 7.53 (s, 1H); 7.55 (s, 1H); 8.06(s, 1H). *MS (EI): 256 (100), 241 (23), 239 (46), 227 (27).

EXAMPLE 4 Preparation of 1-(4-dimethylaminophenyl)-5,6-dihydroxyindane(BXT01049)

5,6-Dihydroxyindan-1-one

5,6-Dimethylindan-1-one (3.13 g; 16.3 mmol) is dissolved in 10 ml ofdichloromethane dried over 3A sieve. A solution of boron tribromide indichloromethane (1M; 28 ml, 28 mmol) is added dropwise at -70° C. tothis light yellow solution. The reaction medium turns claret. Thetemperature of the medium is brought to room temperature, then thesolution is stirred for 2 h 30. The reaction medium is poured onto 50 gof ice, then extracted with ethyl acetate. The organic phases are washedwith saturated aqueous NaCl solution, then dried over MgSO₄. The solventis evaporated under reduced pressure.

The crude product (2.3 g; 87%) is used as it is for the following step.

5,6-Di(tert-butyldimethylsilyloxy)indan-1-one

tert-Butyldimethylsilyl chloride (7.35 g; 49.8 mmol) and imidazole (3.82g; 56.1 mmol) are added to a solution of the 5,6-dihydroxyindan-1-onedescribed above (2 g; 12.2 mmol) in 40 ml of DMF dried over 3A sieve andcooled to 0° C. The temperature is maintained at 0° C. for 10 min, thenat room temperature for 16 h. The product is extracted with ethylacetate. The organic phase is washed with saturated aqueous NaClsolution, then dried over MgSO₄. The solvent is evaporated under reducedpressure. The desired product is purified by chromatography with aneluent: 8:2 hexane/ethyl acetate and is obtained in the form of a beigesolid (4.5 g; 94%).

Physical characteristics:

*¹ H NMR (CDCl₃): 0.195 (s, 6H); 0.205 (s, 6H); 0.92 (s, 18H); 2.60 (t,J=7.63 Hz, 2H); 2.95 (t, J=7.63 Hz, 2H); 6.85 (s, 1H); 7.15 (s, 1H).

5,6-Di(tert-butyldimethylsilyloxy)-3-(4-dimethylaminophenyl)indene

Magnesium (124 mg, 5.1 mmol) is covered with 1 ml of distilled THF andheated at 60° C. with stirring. A solution of4-bromo-N,N-dimethylaniline (1.02 g, 5.1 mmol) in 5 ml of distilled THFis added dropwise over the course of 20 min. The reaction mixture isheated at reflux of the THF for 1 h. A solution of5,6-di(tert-butyldimethylsilyloxy)indan-1-one (2 g, 5.1 mmol) in 10 mlof distilled THF is added dropwise at room temperature over the courseof 5 min to this yellow-green solution. The mixture is brought to refluxfor 20 h. The reaction mixture, which is violet in colour, is hydrolyzedby saturated aqueous ammonium chloride solution, then extracted withethyl acetate. The organic phase is washed with saturated aqueous NaClsolution, then dried over MgSO₄. The solvent is evaporated under reducedpressure. The desired product is purified by chromatography with aneluent: 9:1 hexane/ethyl acetate, and is obtained in the form of an oil(0.9 g; 36%)

Physical characteristics:

*¹ H NMR (CDCl₃): 0.16 (s, 12H); 0.95 (s, 18H); 2.94 (s, 6H); 3.50 (d,J=2.03 Hz, 2H); 6.30 (t, J=2.03 Hz, 1H); 6.75 (m, 2H); 6.95 (s, 1H);7.08 (s, 1H); 7.45 (d, J=7.85 Hz, 2H).

5,6-Di(tert-butyldimethylsilyloxy)-1-(4-dimethylaminophenyl)indane

A solution of the preceding compound (0.9 g; 1.82 mmol) in 10 ml ofmethanol and 2 ml of acetone is stirred in the presence of palladium oncarbon (10%, 60 mg) at room temperature under a hydrogen pressure ofapproximately 5×10⁵ Pa (5 bars) for 3 h. The suspension is filtered,then the filtrate is evaporated to dryness under reduced pressure. Thedesired product is purified by chromatography with an eluent: 9:1hexane/ ethyl acetate, and is obtained in the form of a light beigesolid (0.64 g; 71%).

Physical characteristics:

*¹ H NMR (CDCl₃): 0.08 (s, 6H); 0.18 (s, 6H); 0.90 (s, 9H); 0.95 (s,9H); 1.90 (m, 2H); 2.45 (m, 2H); 2.90 (s, 6H); 4.17 (t, J=8.07 Hz, 1H);6.4 (s, 1H); 6.7 (m, 2H); 7.05 (m, 2H); 7.20 (s, 1H).

5,6-Dihydroxy-1-(4-dimethylaminophenyl)indane

The product obtained previously (0.56 g; 1.12 mmol) is dissolved in 5 mlof distilled THF, then a molar solution of tetrabutylammonium fluoride(2.26 ml; 2.26 mmol) in THF is added with stirring and at roomtemperature. Stirring is maintained for 1 h at this temperature. Afteraddition of water, the product is extracted with ethyl acetate. Theorganic phase is washed with saturated aqueous NaCl solution, then driedover MgSO₄. The solvent is evaporated under reduced pressure. Thedesired product is purified by a double chromatography:

1st purification: eluent: 6:4 hexane-ethyl acetate

2nd purification: eluent: 95:5 dichloromethane-methanol.

The desired product is obtained in the form of a mauve oil (190 mg;62%).

Physical characteristics:

*¹ H NMR (CDCl₃): 1.96 (m, 2H); 2.44 (m, 2H); 2.89 (s, 6H); 4.1 (t,J=7.2 Hz, 1H); 6.4 (s, 1H); 6.73 (d, J=6.7 Hz, 2H); 6.77 (s, 1H); 7.05(d, J=6.7 Hz, 2H). *MS (EI): 269 (100), 254 (10), 240 (9), 225 (28), 77(10).

*HPLC (high performance liquid chromatography): (column: reversed-phaseRP18; length: 15 cm; eluent: 94:6 dichloromethane-methanol; flow rate: 1ml/min, observation wavelength: 254 nm); retention time: 2.37 min;purity: 99.1%.

B. Compounds of Series II EXAMPLE 5 Preparation of2,5,6-trihydroxy-1-(4-hydroxyphenyl)indane (BXT01035)

2-Hydroxy-5,6-dimethoxy-1-(4-methoxyphenyl)indane

A solution of BH₃ THF (1.0M, 4.26 ml, 4.26 mmol) is added at 0° C. overthe course of 15 min to a solution of5,6-dimethoxy-3-(4-methoxyphenyl)indene (1.0 g, 3.55 mmol) in 10 ml ofanhydrous THF. The mixture obtained is stirred for 1 h at the sametemperature and then for 4 h at room temperature. The solution is cooledto 0° C. and 3.5 ml of water are added dropwise, then 7 ml of NaOH (10%strength). A saturated aqueous solution of H₂ O₂ (30% strength, 7 ml) isthen added and the mixture is stirred for 2 h. The mixture is extractedwith 50 ml of AcOEt. The organic phase is washed with 10 ml of water,then dried over MgSO₄. Evaporation of the solvent gives a white solid(1.16 g) which is purified by chromatography, eluting with a 1:3 mixtureof AcOEt/hexane, to give the expected product (white solid, 0.97 g,91%).

Physical characteristics:

m.p.: 128.0°-129.0° C. (AcOEt/hexane=1:4). *¹ H NMR (CDCl₃): 1.96 (d,J=4.68 Hz, 1H, exchangeable with D₂ O); 2.84 (dd, J=6.79-15.39 Hz, 1H);3.21 (dd, J=6.92-15.39 Hz, 1H); 3.73 (s, 3H); 3.79 (s, 3H); 3.87 (s,3H); 4.05 (d, J=6.23 Hz, 1H); 4.39 (m, 1H); 6.46 (s, 1H); 6.81 (s, 1H);6.87 (d, J=8.64 Hz, 2H); 7.07 (d, J=8.64 Hz, 2H). *MS (EI): 300 (100),282 (18), 257 (27), 239 (5), 165 (7), 121 (15).

2,5,6-Trihydroxy-1-(4-hydroxyphenyl)indane

Aluminum tribromide (2.67 g, 10.0 mmol) is mixed with 24 ml ofethanethiol. The preceding phenylindane (300 mg, 1.0 mmol) is added in asingle portion. After 62 h at room temperature, the ethanethiol isevaporated, then the residue is treated with 15 ml of water at 0° C. Themixture is extracted with AcOEt. The organic phases collected are washedwith saturated aqueous NaCl solution, dried over Na₂ SO₄ and evaporatedto dryness to give a yellow oil (250 mg). Purification bychromatography, using a 1:1 mixture of AcOEt/hexane as eluent, gives theexpected product (white solid, 140 mg, 54%).

Physical characteristics:

*¹ H NMR (acetone-d6): 2.68 (dd, J=6.31-15.02 Hz, 1H); 3.06 (dd,J=6.59-15.30 Hz, 1H); 3.90 (d, J=6.53 Hz, 1H); 4.20 (d, J=5.28 Hz, 1H);4.29 (m, 1H); 6.30 (s, 1H); 6.68 (s, 1H); 6.76 (d, J=8.47 Hz, 2H); 6.96(d, J=8.47 Hz, 2H); 7.57 (s, 1H); 7.63 (s, 1H); 8.18 (s, 1H). MS (EI):258 (100), 240 (40), 229 (13), 215 (39), 183 (27), 165 (32), 107 (46),77 (28).

EXAMPLE 6 Preparation of5,6-dihydroxy-1-(4-hydroxyphenyl)-2-methoxyindane (BXT01040)

5,6-Dihydroxy-3-(4-hydroxyphenyl)indene

A solution of BBr₃ in CH₂ Cl₂ (1.0M, 9.60 ml, 9.60 mmol) is addeddropwise to a solution of 5,6-dimethoxy-3-(4-methoxyphenyl)indene (0.60g, 2.23 mmol) in 2 ml of CH₂ Cl₂, cooled to -70° C. The red solutionobtained is heated at room temperature and stirred for 1 h at thistemperature. The reaction mixture is poured into a mixture of ice-waterand AcOEt. The aqueous phase is saturated with NaCl then extracted twicewith AcOEt. The organic phases collected are washed with saturatedaqueous NaCl solution, dried over MgSO₄ and evaporated to dryness togive a red solid (0.62 g) which is used directly in the following step.

Physical characteristics:

*¹ H NMR (acetone-d6): 3.28 (d, J=2.10 Hz, 2H); 6.31 (t, J=2.10 Hz, 1H);6.88 (d, J=8.45 Hz, 2H); 7.03 (s, 1H); 7.07 (s, 1H); 7.44 (d, J=8.45 Hz,2H); 7.72 (broad s, 2H); 8.53 (broad s, 1H). *MS (EI): 240 (96), 222(7), 194 (32), 181 (15), 165 (27), 152 (14), 43 (100).

5,6-Dibenzyloxy-1-(4-benzyloxyphenyl)indene

The crude product from the preceding step (0.62 g) is dissolved in 8 mlof acetone. K₂ CO₃ (1.85 g, 13.38 mmol) and benzyl bromide (1.73 g, 10.0mmol) are added thereto. The mixture is heated at reflux for 18 h. Thesolid is filtered off, then the filtrate is evaporated to dryness. Theresidue is chromatographed directly, eluting with a 1:5 mixture ofAcOEt/hexane, to give the expected product (0.82 g) which is useddirectly in the following step.

Physical characteristics:

*¹ H NMR (CDCl₃): 3.36 (d, J=1.83 Hz, 2H); 5.11 (s, 2H); 5.13 (s, 2H);5.17 (s, 2H); 6.37 (t, J=1.83 Hz, 1H); 7.01 (d, J=8.72 Hz, 2H); 7.17 (s,1H); 7.25-7.60 (m, 18H).

5,6-Dibenzyloxy-1-(4-benzyloxyphenyl)-2-hydroxyindane

A solution of BH₃.THF (1.0M, 3.20 ml, 3.20 mmol) is added at roomtemperature over the course of 15 min to a solution of the abovephenylindene (0.82 g, 1.61 mmol) in 8 ml of anhydrous THF. The mixtureobtained is stirred for 3.5 h at room temperature. The solution iscooled to 0° C. and 3.5 ml of water are added dropwise, then 7 ml ofNaOH (10% strength). An aqueous solution of H₂ O₂ (30% strength, 7 ml)is then added and the mixture is stirred for 2 h. The mixture isextracted with AcOEt. The organic phase is washed with water, then driedover MgSO₄. Evaporation of the solvent gives a colourless oil (0.95 g)which is purified by chromatography, eluting with a 1:4 mixture ofAcOEt/hexane, to give the expected product (white solid, 0.46 g, 54%).

Physical characteristics:

*¹ H NMR (CDCl₃): 2.02 (broad s, exchangeable with D₂ O, 1H); 2.81 (dd,J=7.00-15.26 Hz, 1H); 3.17 (dd, J=6.64-15.26 Hz, 1H); 4.03 (d, J=6.28Hz, 1H); 4.34 (m, 1H); 5.01 (s, 2H); 5.06 (s, 2H); 5.15 (s, 2H): 6.57(s, 1H); 6.86 (s, 1H); 6.92 (d, J=8.72 Hz, 2H); 7.40 (d, J=8.72 Hz, 2H);7.24-7.50 (m, 15H). MS (CI, NH₃): 546 (MNH₄ ⁺).

5,6-Dibenzyloxy-1-(4-benzyloxyphenyl)-2-methoxyindane

The preceding alcohol (0.26 g, 0.49 mmol) is mixed with potassium powder(0.11 g, 1.97 mmol). 3 ml of DMSO are added thereto, followedimmediately by methyl iodide (0.14 g, 9.85 mmol). The mixture is stirredat room temperature for 3 h. 5 ml of water are added. The mixture isextracted with AcOEt. The organic phases collected are washed withsaturated aqueous NaCl solution, dried over MgSO₄ and evaporated to givea yellow oil. The purification of this oil by chromatography (eluent:AcOEt/hexane=1:9) gives the expected product (0.26 g, 97%).

Physical characteristics:

*¹ H NMR (CDCl₃): 2.85 (dd, J=7.01-15.35 Hz, 1H); 3.23 (dd, J=6.78-15.35Hz, 1H); 3.38 (s, 3H); 4.04 (m, 1H); 4.12 (d, J=6.25 Hz, 1H); 5.03 (s,2H); 5.08 (s, 2H); 5.14 (s, 2H); 6.60 (s, 1H); 6.88 (s, 1H); 6.93 (d,J=8.64 Hz, 2H); 7.10 (d, J=8.64 Hz, 2H); 7.30-7.60 (m, 15H).

5,6-Dihydroxy-1-(4-hydroxyphenyl)-2-methoxyindane

A solution of the preceding compound (0.26 g, 0.48 mmol) in 10 ml ofAcOEt is hydrogenated under approximately 10⁵ Pa (1 atm) of hydrogen inthe presence of palladium on carbon (10%, 10 mg). The reaction isfinished after 12 h. The catalyst is filtered off, then the solvent isevaporated under reduced pressure to give a yellow oil. This crudeproduct is purified by chromatography (eluent: AcOEt/hexane=1:4) to givethe expected product (white solid: 92 mg, 71%).

Physical characteristics:

*¹ H NMR (acetone-d6): 2.68 (dd, J=6.15-15.62 Hz, 1H); 3.15 (dd,J=6.25-15.62 Hz, 1H); 3.24 (s, 3H); 4.01 (m, 1H); 4.04 (d, J=6.25 Hz,1H); 6.29 (s, 1H); 6.70 (s, 1H); 6.76 (d, J=8.24 Hz, 2H); 6.98 (d,J=8.24 Hz, 2H), 7.62 (broad s, 2H); 8.17 (broad s, 1H). *MS (EI): 272(100), 240 (81), 227 (18).

C. Compounds of Series III EXAMPLE 7 Preparation of5,6,6a,11b-tetrahydro-3,9,10-trihydroxybenzo[c]fluorene (BXT01050)

2-(3,4-Dimethoxybenzylidene)-6-methoxy-1-tetralone

6-Methoxy-1-tetralone (7.04 g, 40 mmol) and 3,4-dimethoxybenzaldehyde(6.44 g, 40 mmol) are mixed with 100 ml of absolute ethanol. The mixtureis stirred for 0.5 h to dissolve as much as possible of the reagents.The introduction of gaseous hydrogen chloride is commenced. Anexothermic reaction is noted which enables the complete dissolution ofall of the reagents. The round-bottomed flask is cooled from time totime with an ice bath so that the temperature does not exceed 80° C. Themixture is thus saturated with hydrogen chloride after 1 h to give anintense red color. This mixture is allowed to stand at room temperaturefor 2 h. The reaction mixture is poured into a mixture of 400 ml ofwater and 40 ml of aqueous NaOH (3N). It is stirred vigorously for 3 hat room temperature. The precipitated solid is filtered over a glassfrit, washed with water and finally dried under vacuum, over P₂ O₅, togive a solid (12.35 g, 95%). A small quantity of product isrecrystallized from the mixture AcOEt/hexane=1:2.

Physical characteristics:

*¹ H NMR (CDCl₃): 2.89 (t, J=6.96 Hz, 2H); 3.12 (dt, J=1.70-6.96 Hz,2H), 3.85 (s, 3H); 3.89 (s, 3H); 3.90 (s, 3H); 6.68 (d, J=2.48 Hz, 1H);6.85 (dd, J=2.48-8.67 Hz, 1H); 6.88 (d, J=8.14 Hz, 1H); 6.95 (d, J=1.64Hz, 1H); 7.04 (dd, J=1.64-8.14 Hz, 1H); 7.78 (s, 1H); 8.08 (d, J=8.67Hz, 1H). *MS (EI): 324 (87), 323 (100), 309 (38), 293 (39), 281 (11).*m.p.: 109.0°-110.0° C. (AcOEt/hexane=1:2).

2-(3,4-Dimethoxybenzyl)-6-methoxy-1-tetralone

2.0 g of palladium on carbon (10%) are added to a solution of thepreceding compound (35.0 g, 0.107 mol) in 550 ml of AcOEt. The mixtureis hydrogenated under approximately 2×10⁵ Pa (2 atm) of hydrogen for 1h. The catalyst is removed by filtration and the filtrate is evaporatedto dryness to give 38.4 g of a crude product which is dissolved in 100ml of hot AcOEt. 130 ml of hexane are added. The crystals formed (23.56g) are filtered. The filtrate is evaporated to dryness. The residue ischromatographed, eluting with a 1:4 mixture of AcOEt/hexane, to give6.03 g of the pure product expected.

Yield: 29.59 g (84%).

Physical characteristics:

*m.p.: 101.0°-102.0° C. (AcOEt/hexane=1:2). *¹ H NMR (CDCl₃): 1.67-1.83(m, 1H); 2.00-2.14 (m, 1H); 2.51-2.72 (m, 2H); 2.84-2.96 (m, 2H);3.34-3.48 (m, 1H); 3.82 (s, 3H); 3.84 (s, 3H); 3.85 (s, 3H); 6.64 (d,J=2.36 Hz, 1H); 6.74-6.84 (m, 4H); 8.02 (d, J=8.74 Hz, 1H). MS (EI): 326(35), 175 (9), 151 (100), 107 (13), 91 (17), 77 (11).

5,6-Dihydro-3,9,10-trimethoxybenzo[c]fluorene

15.0 g of polyphosphoric acid are mixed with the tetralone obtainedpreviously (1.04 g, 3.2 mmol), and are then heated at 80°-90° C. for 4h. After having cooled the mixture to room temperature, 10 g ofice-water and 20 ml of AcOEt are added. The organic phase is separatedand the aqueous phase is extracted with AcOEt. The combined organicphases are washed with saturated aqueous NaHCO₃ solution then with NaCl(sat.), dried over MgSO₄ and evaporated to dryness to give a solid (0.91g) which is purified by chromatography, eluting with a 1:6 mixture ofAcOEt/hexane. The expected product (0.61 g) and the starting product(0.24 g) are obtained. the yield of this reaction in relation to theproduct consumed is 81%.

Physical characteristics:

*¹ H NMR (CDCl₃): 2.59 (t, J=7.77 Hz, 2H); 2.89 (t, J=7.77 Hz, 2H); 3.39(s, 2H); 3.84 (s, 3H); 3.90 (s, 3H); 3.94 (s, 3H); 6.84 (m, 2H); 7.08(s, 1H); 7.38 (s, 1H); 7.72 (d, J=9.24 Hz, 1H). *m.p.: 146.5°-147.0° C.(AcOEt/hexane=1:4). *MS (EI): 308 (100), 293 (14), 277 (38), 202 (24),176 (33), 165 (17), 152 (21).

5,6,6a,11b-Tetrahydro-3,9,10-trimethoxybenzo[c]fluorene

0.15 g of palladium on carbon (10%) is added to a solution of thepreceding product (0.90 g, 2.92 mmol) in 30 ml of AcOEt. The mixture ishydrogenated under a pressure of approximately 8×10⁵ Pa (8 atm) ofhydrogen for 5 h. The catalyst is removed by filtration and the filtrateis evaporated to dryness to give the pure product expected (0.90 g,99%).

Physical characteristics:

*m.p.: 77.0°-79.0° C. (AcOEt/hexane=1:4). *¹ H NMR (CDCl₃): 1.45-1.65(m, 1H); 1.66-1.80 (m, 1H); 2.75 (dd, J=6.96-15.10 Hz, 1H); 2.68-2.87(m, 3H); 3.15 (dd, J=6.96-15.10 Hz, 1H); 3.79 (s, 3H); 3.80 (s, 3H);3.84 (s, 3H); 4.21 (d, J=6.68 Hz, 1H); 6.66 (d, J=2.64 Hz, 1H); 6.76 (s,1H); 6.78 (s, 1H); 6.83 (dd, J=2.64-8.38 Hz, 1H); 7.33 (d, J=8.38 Hz,1H). MS (EI): 310 (100), 295 (23), 279 (46), 267 (15), 189 (24), 121(23).

5,6,6a,11b-Tetrahydro-3,9,10-trihydroxybenzo[c]fluorene

A solution of BBr₃ in CH₂ Cl₂ (1.0M, 6.5 ml, 6.5 mmol) is added dropwiseto a solution of the compound obtained previously (0.57 g, 1.84 mmol) in4 ml of CH₂ Cl₂, cooled to -70° C. The red solution obtained is heatedto room temperature and stirred for 0.5 h at this temperature. Thereaction mixture is poured into a mixture of ice-water and AcOEt. Theaqueous phase is saturated with NaCl, then extracted twice with AcOEt.The organic phases collected are washed with saturated aqueous NaClsolution, dried over MgSO₄ and evaporated to dryness to give a yellowoil (0.50 g). Chromatography of 0.28 g of this oil (eluent:AcOEt/hexane=1:1) enables the isolation of the expected compound (0.24g, 87%).

Physical characteristics:

*¹ H NMR (acetone-d6): 1.46 (m, 1H); 1.61 (m, 1H); 2.46-2.81 (m, 4H);3.01 (dd, J=7.00-14.98 Hz, 1H); 4.05 (d, J=6.68 Hz, 1H); 6.54 (d, J=2.54Hz, 1H); 6.66 (s, 1H); 6.68 (s, 1H); 6.71 (dd, J=2.54-8.34 Hz, 1H); 7.18(d, J=8.34 Hz, 1H); 7.50 (broad s, 2H); 7.83 (broad s, 1H). *MS (EI):268 (82), 251 (31), 240 (100), 161 (21), 145 (26), 77 (30).

EXAMPLE 8 Preparation of5,6,6a,7,8,12b-hexahydro-2,3,10-trihydroxybenzo[c]phenanthrene(BXT01051)

2'-(3,4-Dimethoxyphenyl)tetral-6-methoxy-2-spiro-1'-cyclopropan-1-one

10 ml of DMSO are added to a mixture of trimethylsulphoxonium iodide(4.40 g, 20 mmol) and sodium hydride (80% strength in mineral oil, 0.80g, 20 mmol). The mixture is stirred at room temperature for 1.5 h. 30 mlof DMSO are added and then, in small portions, the2-(3,4-dimethoxybenzylidene)-6-methoxy-1-tetralone described previouslyis added over the course of 15 min. The mixture is stirred for 18 h atroom temperature and then hydrolyzed by the addition of 50 ml ofsaturated aqueous NH₄ Cl solution. The mixture is extracted with AcOEt.The organic phases collected are washed with water, dried over MgSO₄ andevaporated under reduced pressure to give a colorless oil (7.0 g).Purification of this oil by chromatography, eluting with a 1:4 mixtureof AcOEt/hexane, gives the desired product (5.41 g, 85%).

Physical characteristics:

*¹ H NMR (CDCl₃): 1.26 (dd, J=4.24-7.08 Hz, 1H); 1.68 (m, 1H); 1.85 (m,2H); 2.70 (m, 2H); 2.94 (dd, J=7.08-8.24 Hz, 1H); 3.83 (s, 3H); 3.85 (s,3H); 3.86 (s, 3H); 6.64 (d, J=2.52 Hz, 1H); 6.77 (m, 3H); 6.82 (dd,J=2.52-8.50 Hz, 1H); 8.00 (d, J=8.72 Hz, 1H). *MS (EI): 338 (100), 323(12), 200 (20), 176 (19), 151 (73), 91 (29), 77 (51).

2-(3,4-Dimethoxyphenylethyl)-6-methoxy-1-tetralone

A solution of the cyclopropane derivative obtained previously (1.0 g,2.96 mmol) and para-toluene sulphonic acid (300 mg) in 20 ml of CH₂ Cl₂is stirred at room temperature for 5 h. This solution is then washedwith saturated aqueous NaHCO₃ solution, dried over MgSO₄ and evaporatedunder reduced pressure to give a yellow oil (1.05 g). The purificationof this product by chromatography (eluent: AcOEt/hexane=1:4) gives asolid (0.57 g) which is a mixture of the two isomeric alkenes.

0.53 g of this latter mixture is taken up in 15 ml of AcOEt. Thesolution is hydrogenated in the presence of palladium on carbon (10%, 50mg) under a pressure of approximately 2×10⁵ Pa (2 bars) of hydrogen for1 h. The catalyst is filtered and the filtrate is evaporated underreduced pressure to give the expected product (0.53 g, 57%).

Physical characteristics:

*¹ H NMR (CDCl₃): 1.80 (m, 2H); 2.24 (m, 2H); 2.42 (m, 1H), 2.66 (m,2H); 2.92 (m, 2H); 3.81 (s, 3H); 3.82 (s, 3H); 3.84 (s, 3H); 6.64 (d,J=2.36 Hz, 1H); 6.77 (m, 3H); 6.79 (dd, J=2.36-8.80 Hz, 1H); 7.97 (d,J=8.72 Hz, 1H). *MS (EI): 340 (2.5), 176 (100), 164 (16), 151 (12).

5,6,7,8-Tetrahydro-2,3,10-trimethoxybenzo[c]phenanthrene

8 g of polyphosphoric acid are mixed with the tetralone obtainedpreviously (0.41 g, 1.21 mmol), then heated at 80°-90° C. for 2.5 h.After having cooled the mixture to room temperature, 20 g of ice-waterand 20 ml of AcOEt are added. The organic phase is removed afterallowing the medium to settle and the aqueous phase is extracted withAcOEt. The combined organic phases are washed with saturated aqueousNaHCO₃ solution, then with saturated aqueous NaCl solution, dried overMgSO₄ and evaporated to dryness to give a solid (0.38 g) which ispurified by chromatography, eluting with a 1:9 mixture of AcOEt/hexane.The expected product (0.19 g; 49%) is obtained.

Physical characteristics:

¹ H NMR (CDCl₃): 2.31 (m, 4H); 2.66 (m, 4H); 3.80 (s, 3H); 3.82 (s, 3H);3.89 (s, 3H); 6.77 (m, 3H); 7.08 (s, 1H); 7.44 (d, J=8.42 Hz, 1H). MS(EI): 322 (100), 307 (10), 291 (19), 189 (18), 161 (18).

5,6,6a,7,8,12b-Hexahydro-2,3,10-trimethoxybenzo[c]phenanthrene

20 mg of palladium on carbon (10%) are added to a solution of thepreceding product (170 mg, 0.50 mmol) in 10 ml of AcOEt. The mixture ishydrogenated under a pressure of approximately 8×10⁵ Pa (8 atm) for 2.5h. The catalyst is removed by filtration and the filtrate is evaporatedto dryness to give an oil. This oil is purified by chromatography,eluting with a 1:9 mixture of AcOEt/hexane, to give the desired product(150 mg, 88%).

Physical characteristics:

*¹ H NMR (CDCl₃): 1.53 (m, 2H); 1.81 (m, 1H); 2.00 (m, 1H); 2.32 (m,1H); 2.75 (m, 4H); 3.77 (s, 3H); 3.78 (s, 3H); 3.81 (1H, hidden by theOMe's); 3.85 (s, 3H); 6.65 (m, 4H); 6.99 (d, J=9.16 Hz, 1H). MS (EI):324 (100), 309 (5), 296 (33), 293 (37), 203 (36), 189 (86), 173 (39).

5,6,6a,7,8,12b-Hexahydro-2,3,10-trihydroxybenzo[c]phenanthrene

A solution of BBr₃ in CH₂ Cl₂ (1.0M, 1.5 ml, 1.5 mmol) is added dropwiseto a solution of the derivative obtained previously (150 mg, 0.37 mmol)in 1 ml of CH₂ Cl₂, cooled to -70° C. The red solution obtained isheated to room temperature and stirred for 0.5 h at this temperature.The reaction mixture is poured into a mixture of ice-water and AcOEt.The aqueous phase is saturated with NaCl, then extracted twice withAcOEt. The combined organic phases are washed with saturated aqueousNaCl solution, dried over MgSO₄ and evaporated to dryness to give ayellow oil (0.18 g). Chromatography of this oil (eluent:AcOEt/hexane=1:1) enables the isolation of the expected compound (91 mg,87%).

Physical characteristics:

*¹ H NMR (acetone-d6): 1.53 (m, 2H); 1.90 (m, 2H); 2.32 (m, 1H); 2.75(m, 4H); 3.67 (d, J=4.76 Hz, 1H); 6.48 (s, 1H); 6.55 (s, 1H), 6.59 (m,2H); 6.88 (d, J=8.88 Hz, 1H); 7.51 (broad s, 2H); 7.95 (broad s, 1H).*MS (EI): 282 (100), 265 (16), 254 (53), 175 (26), 161 (36), 115 (31),91 (22), 77 (24).

D. Compounds of Series IV EXAMPLE 9 Preparation of 3-O-methylbrazilin

5',6',7-Tri-O-benzylbrazilin:

A solution of brazilin monohydrate (304 mg; 1.0 mmol; ALDRICH) inacetone (2.5 ml) containing benzyl bromide (0.4 ml; 3.36 mmol; JANSSEN)and anhydrous potassium carbonate (1.06 g; 7.68 mmol; OSI), is heated atreflux under an inert atmosphere for 15 h. The reaction mixture isviolet in color. A solution of sodium hydroxide (5 ml, 0.5N) is added,and the solution thus obtained is stirred for 1 h at room temperature.The reaction medium is extracted with ethyl acetate (2×10 ml). Theorganic phases are combined, washed with water (2×5 ml) and dried overMgSO₄. The solvent is evaporated under reduced pressure. The desiredproduct is purified by chromatography on a MERCK F254 silica column(eluent=hexane/AcOEt, 3:1). Yield=530 mg (95.3%).

Physical characteristics:

*¹ H NMR (200 MHz, CDCl₃): 2.45 (s, 1H); 2.83 (d, 1H, J=16.0 Hz); 3.18(d, 1H, J=16.0 Hz); 3.78 (d, 1H, J=11.3 Hz); 4.02 (d, 1H, J=11.3 Hz);4.09 (s, 1H); 5.04 (s, 2H); 5.09 (s, 2H); 5.11 (s, 2H); 6.55 (d, 1H,J=2.6 Hz); 6.71 (dd, 1H, J=2.6-8.5 Hz); 6.80 (s, 1H); 6.88 (s, 1H); 7.17(d, 1H, J=8.5 Hz); 7.42 (m, 15H). *MS (EI): (M⁺)=556 (2), 466 (1), 181(2), 108 (3), 91 (100).

5',6',7-Tri-O-benzyl-3-O-methylbrazilin:

Potassium powder (210 mg; 3.80 mmol) is introduced under an inertatmosphere at room temperature into a solution of5',6',7-tri-O-benzylbrazilin (530 mg; 0.95 mmol) in dry DMSO (4 ml).Methyl iodide (0.12 ml; 1.90 mmol) is then added to the reaction mediumwhich is stirred for 30 minutes under these conditions. After additionof water (5 ml), the solution is extracted with ethyl acetate (15 ml).The organic phase is dried over MgSO₄. The solvent is evaporated underreduced pressure. The desired product is purified by chromatography on aMERCK F254 silica column (eluent=hexane/AcOEt, 2:1). Yield=510 mg (94%).

The product is recrystallized from a 3:1 mixture of hexane/AcOEt.

Physical characteristics:

*m.p.=99.5°-100.5° C. *¹ H NMR (200 MHz, CDCl₃): 2.70 (d, 1H, J=15.8Hz); 3.22 (d, 1H, J=15.8 Hz); 3.38 (s, 3H); 3.67 (d, 1H, J=11.9 Hz);4.20 (s, 1H); 4.26 (d, 1H, J=11.9 Hz), 5.03 (s, 2H); 5.07 (s, 2H); 5.11(s, 2H); 6.54 (d, 1H, J=2.6 Hz); 6.68 (dd, 1H, J=2.6-8.5 Hz); 6.78 (s,1H); 6.88 (s, 1H); 7.17 (d, 1H, J=8.5 Hz); 7.42 (m, 15H). *MS (EI):(M⁺)=570 (45), (M⁺ -91)=479 (12), 91 (100).

3-O-Methylbrazilin:

A solution of 5',6',7-tri-O-benzyl-3-O-methylbrazilin (160 mg; 0.28mmol)in a 5:2 mixture of AcOEt/MeOH, containing 10% palladium on carbon (20mg), is purged for 15 minutes by a stream of hydrogen. The suspension ishydrogenated for 5 h at room temperature with stirring. It issubsequently filtered under an inert atmosphere. The solvent isevaporated under reduced pressure. The desired product graduallycrystallizes. Yield=81 mg (96%).

Physical characteristics:

*¹ H NMR (200 MHz, CD₃ COCD₃): 2.68 (d, 1H, J=15.6 Hz); 3.08 (d, 1H,J=15.6 Hz); 3.28 (s, 3H); 3.52 (d, 1H, J=12.1 Hz); 4.03 (s, 1H); 4.24(d, 1H, J=12.1 Hz); 6.28 (d, 1H, J=2.5 Hz); 6.48 (dd, 1H, J=2.5-8.5 Hz);6.64 (s, 1H); 6.71 (s, 1H); 7.18 (d, 1H, J=8.5 Hz); 7.79 (s, 1H); 7.85(s, 1H); 8.43 (s, 1H). *MS (EI): 300 (5), 268 (4), 110 (6), 84 (41), 66(49).

EXAMPLE 10 Preparation of brazilane (R¹ =OH; R² =--CH₂ --O--; R³ =H)

5',6',7-Tri-O-benzyl-3-O-phenyloxythionocarbonylbrazilin:

A solution of methyllithium (0.4 ml of a 1.6M solution in cyclohexane,JANSSEN) is added dropwise, under nitrogen and with stirring, to asolution of 5',6',7-tri-O-benzylbrazilin (280 mg, 0.50 mmol) in 3 ml ofTHF, cooled to -78° C. Stirring is maintained for 10 minutes at thistemperature then for 1 hour at room temperature. The reaction mixture isagain cooled to -78° C. and phenyl chlorothionocarbonate (0.21 ml, 0.75mmol, ALDRICH) is added dropwise. The reaction mixture is allowed toreturn to room temperature and is stirred for 1 hour. Hydrolysis iscarried out using ammonium chloride, then the product is extracted withethyl acetate (2×10 ml). The organic phases are combined, washed withsaturated sodium chloride solution (2×10 ml), then dried over MgSO₄. Thesolvent is evaporated under vacuum. The yellow oil thus obtained ischromatographed on a MERCK F254 silica column. The desired product,eluted with a 1:5 mixture of ether/hexane, is obtained with a yield of83% (280 mg of a colorless solid).

Physical characteristics:

*¹ H NMR (200 MHz, CDCl₃): 3.51 (d, 1H, J=16.50 Hz); 3.71 (d, 1H,J=16.50 Hz); 3.75 (d, 1H, J=12.17 Hz); 4.58 (s, 1H); 5.05 (s, 4H); 5.09(s, 2H); 5.27 (d, 1H, J=12.17 Hz); 6.57 (d, 1H, J=2.56 Hz); 6.69 (dd,1H, J=2.56-8.45 Hz); 6.78 (s, 1H); 6.88 (s, 1H); 7.06 (d, 2H, J=7.30Hz); 7.19 (d, 1H, J=8.45 Hz); 7.24-7.46 (m, 18H). *MS (EI): (M⁺-PhOC(OH)═S)=538 (3); 447 (1.5); 419 (3); 91 (100); 60 (80).

5',6',7-Tri-O-benzylanhydrobrazilin

A solution of the thionocarbonate derivative previously obtained (100mg, 0.15 mmol) in 3 ml of dry toluene is heated at reflux for 2 hours.The solvent of the slightly yellowish solution thus obtained isevaporated under vacuum. The residue is purified by chromatography on aMERCK F254 silica column and eluted with a 1:5 mixture of ether/hexane.The desired product is obtained with a yield of 82% (64 mg).

Physical characteristics:

*m.p.=142°-144° C. (ethyl acetate/hexane=1:4). *¹ H NMR (200 MHz,CDCl₃): 3.30 (s, 2H); 5.05 (s, 2H); 5.12 (s, 2H); 5.17 (s, 2H); 5.21 (s,2H); 6.58 (s, 1H); 6.60 (dd, 1H, J=2.50-8.41 Hz); 7.13 (s, 1H);7.30-7.50 (m, 17H). *MS (EI): 538 (12), 447 (3), 419 (6), 91 (100).

Brazilane:

A solution of the preceding derivative (92 mg, 0.17 mmol) in 5 ml ofethyl acetate is hydrogenated in the presence of palladium on carbon(10%, 30 mg, ALDRICH) under a pressure of approximately 8×10⁵ Pa (8bars) for 3 hours at room temperature. The suspension is filtered andthe solvent is evaporated under vacuum. The desired product is obtainedafter purification by chromatography on a MERCK F254 silica column withan eluent of 1:1 ether/hexane. Yield=69%.

Physical characteristics:

*¹ H NMR (200 MHz, CD₃ COCD₃): 2.47 (dd, 1H, J=2.38-15.46 Hz); 2.80 (m,1H); 3.03 (dd, 1H, J=7.32-15.46 Hz); 3.47 (t, 1H, J=10.62 Hz); 4.02 (dd,1H, J=4.48-10.62 Hz); 4.06 (d, 1H, J=7.34 Hz); 6.25 (d, 1H, J=2.52 Hz);6.45 (dd, 1H, J=2.52-8.34 Hz); 6.67 (s, 1H); 6.80 (s, 1H); 7.20 (d, 1H,J=8.34 Hz); 7.55 (s, 1H); 7.61 (s, 1H); 8.15 (s, 1H). *MS (EI): 270(100), 253 (31), 240 (18), 205 (22), 161 (39), 115 (25), 101 (24).

E. Compound of Series V EXAMPLE 11 Preparation of 7-O-methylbrazilin

5',6'-O-Methylenebrazilin:

A solution of anhydrous potassium fluoride (145 mg, 2.5 mmol, JANSSEN)and brazilin monohydrate (152 mg, 0.5 mmol, ALDRICH) in 1 ml ofdimethylformamide is heated and stirred under nitrogen at 120° C.Dibromomethane (39 μl, 0.55 mmol, JANSSEN) is added. After stirring atthis temperature for 18 hours, the reaction mixture is cooled to 0° C.and 4 ml of water are added. The mixture is extracted with ethyl acetate(2×5 ml). The organic phases are combined, washed with water and thenwith saturated aqueous sodium chloride solution and dried over Na₂ SO₄.The solvent is evaporated under vacuum. The desired product is obtainedafter purification by chromatography on a MERCK F254 silica column(eluent=ethyl acetate/hexane, 1:1). Yield: 42%

The product is recrystallized from a 1:1 mixture of ethylacetate/hexane.

Physical characteristics:

*m.p.=264.5° C. (decomposition). *¹ H NMR (200 MHz, DMSO d₆): 2.81 (d,1H, J=16.3 Hz); 2.86 (d, 1H, J=16.3 Hz); 3.64 (d, 1H, J=11.8 Hz); 3.82(d, 1H, J=11.8 Hz); 3.88 (s, 1H); 5.36 (s, 1H); 5.91 (s, 1H); 5.93 (s,1H); 6.18 (d, 1H, J=2.4 Hz); 6.40 (dd, 1H, J=2.4-8.2 Hz); 6.74 (s, 1H);6.87 (s, 1H); 7.22 (d, 1H, J=8.20 Hz); 9.26 (s, 1H). *SM (EI): 298(100), 280 (62), 279 (79), 241 (28), 149 (58).

7-O-Methyl-5',6'-O-methylenebrazilin:

Potassium carbonate (183 mg, 1.33 mmol, OSI) and methyl iodide (35 μl,0.53 mmol, LANCASTER) are added to a solution of5',6'-O-methylenebrazilin (79 mg, 0.27 mmol) in 2 ml of acetone. Themixture is heated at reflux under nitrogen for 24 hours with stirring.Then, after returning to room temperature, the suspension is filtered.The filtrate is evaporated to dryness and the desired product isobtained after purification by chromatography on a MERCK F254 silicacolumn (eluent=ethyl acetate/hexane, 1:4). Yield=65%.

Physical characteristics:

*¹ H NMR (200 MHz, CDCl₃): 2.58 (s, 1H); 2.80 (d, 1H, J=16.45 Hz); 3.17(d, 1H, J=16.45 Hz); 3.76 (s, 3H); 3.79 (d, 1H, J=11.8 Hz); 4.00 (d, 1H,J=16.45 Hz); 4.04 (s, 1H); 5.86 (m, 2H); 6.48 (d, 1H, J=2.4 Hz); 6.62(dd, 1H, J=2.4-8.2 Hz); 6.68 (s, 1H); 6.72 (s, 1H); 7.24 (d, 1H, J=8.20Hz).

7-O-Methylbrazilin:

A solution of boron trichloride (0.25 ml of a 1M solution in methylenechloride, 0.25 mmol) is added dropwise, under nitrogen and withstirring, to a solution of 7-O-methyl-5',6'-O-methylenebrazilin (50 mg,0.16 mmol) in 2 ml of methylene chloride. The reaction mixture isstirred at room temperature for 20 hours. Absolute methanol (60 μl) isslowly added, then the solvent is evaporated under vacuum. The residueis taken up in ethyl acetate (5 ml) and washed with water. The organicphase is dried over Na₂ SO₄ and the solvent is evaporated under vacuum.The desired product is obtained after purification by chromatography ona MERCK F254 silica column (eluent=ethyl acetate/hexane, 1:1). Yield=44%.

Physical characteristics:

*¹ H NMR (200 MHz, CD₃ COCD₃): 2.80 (d, 1H, J=16.5 Hz); 3.00 (d, 1H,J=16.50 Hz); 3.70 (s and d, 4H); 3.93 (d, 1H, J=11.30 Hz); 3.98 (s, 1H);4.48 (s, 1H); 6.37 (d, 1H, J=2.4 Hz); 6.56 (dd, 1H, J=2.4-8.30 Hz); 6.63(s, 1H); 6.77 (s, 1H); 7.26 (d, 1H, J=8.30 Hz); 7.59 (s, 1H); 7.64 (s,1H). *MS (EI): 300 (100), 282 (59), 281 (63), 243 (45), 176 (13), 165(10), 141 (20).

II. Preparation of compounds of general formula II("mercaptan-scavenging reagents") EXAMPLE 12 Preparation of1,4,6-trimethyl-2-vinylpyridinium tetrafluoroborate

4,6-Dimethyl-2-vinylpyridine:

Benzoyl chloride (29.12 g; 0.186 mol; JANSSEN) in 100 ml of THF is addedto a solution of 2,4-dimethylpydirine (20 g; 0.186 mol; LANCASTER) in200 ml of freshly distilled THF, cooled to -20° C. and under an inertatmosphere. The reaction mixture is stirred at this temperature for 20minutes. Then vinylmagnesium bromide (186 ml of a 1.0M solution in THF)is added and the solution is stirred at room temperature for 0.5 h. Thesolvent is evaporated under reduced pressure and the residue is taken upin 300 ml of methyl tert-butyl ether. After washing the reaction mediumwith saturated NH₄ Cl solution, the organic phase is dried over MgSO₄and the desired product is purified by chromatography on a MERCK F254silica column (eluent=hexane/AcOEt, 9:1). Yield=39.5 g (83%), colorlessoil.

The preceding product is taken up in 500 ml of toluene, and p-chloranil(tetrachloro-1,4-benzoquinone) (41.7 g; 0.169 mol) is added, and themixture is then heated at reflux for 2 h. The reaction medium isconcentrated under reduced pressure, then sodium hydroxide solution (260ml, 2N) is added. The product is extracted with methyl tert-butyl ether(300 ml) and purified by chromatography on a MERCK F254 silica column(eluent=hexane/AcOEt, 9:1). Yield=11.1 g (54.2%), pale yellow oil.

Physical characteristics:

*¹ H NMR (200 MHz, CD₃ COCD₃): 2.20 (s, 3H); 2.50 (s, 3H); 5.25-5.45(dd, 2H, J=11.9-18.7 Hz); 6.60-6.90 (m, 3H). *MS (CI, NH₃): MH⁺ =134(100).

1,4,6-Trimethyl-2-vinylpyridinium tetrafluoroborate:

Trimethyloxonium tetrafluoroborate (0.50 g; 3.76×10⁻³ mol) is added inone portion to a solution of 4,6-dimethyl-2-vinylpyridine (0.61 g;4.13×10⁻³ mol) in 4.0 ml of methylene chloride under an inertatmosphere. The suspension is left to react for 2 h at 25° C. Thesuspension is filtered and the product is dried under vacuum.Yield=0.162 g (18%), white crystals.

Physical characteristics:

*¹ H NMR (200 MHz, CD₃ COCD₃): 2.60 (s, 3H); 2.85 (s, 3H); 4.20 (s, 3H);6.00 (d, 1H, J=12.8 Hz); 6.25 (d, 1H, J=19.2 Hz); 7.27 (dd, 1H,J=12.8-19.2 Hz); 7.82 (s, 1H); 7.95 (s, 1H). *MS (EI): (M⁺) 148 (10);147 (18); 133 (34); 121 (100); 106 (25); 91 (31); 77 (52); 49 (77).

*Microanalysis: Calculated (%) : C 51.10; H 6.00; N 6.00 Found (%): C49.80; H 6.08; N 5.96.

EXAMPLE 13 Preparation of 1-methyl-4-vinylquinolinium tetrafluoroborate

4-Vinylquinoline:

A solution of 4-quinolinecarboxaldehyde (5 g; 3.18×10⁻² mol) in 32 ml ofbenzene, containing triphenylmethylphosphonium iodide (26 g; 6.36×10⁻²mol), is added under an inert atmosphere to sodium hydroxide solution(96 ml; 5N). The two-phase system is stirred at 45° C. for 30 minutes.After allowing the phases to separate by settling, the aqueous phase isextracted with ethyl acetate (3×100 ml). The organic phases are combinedand washed with 1N HCl solution (2×30 ml), then the aqueous phase isneutralized with NaHCO₃ and extracted with methylene chloride (3×100ml). The organic phase is dried over MgSO₄. The desired product ispurified by chromatography on a MERCK F254 silica column(eluent=cyclohexane/AcOEt, 8:2). Yield=2.8 g (60%).

Physical characteristics:

¹ H NMR (200 MHz, CDCl₃): 5.60(d, 1H, J=10 Hz); 5.95 (d, 1H, J=14 Hz);7.30-7.55 (m, 4H); 7.60-7.75 (t, 1H, J=4 Hz); 8.07 (t, 2H, J=6 Hz); 8.85(d, 1H, J=4 Hz).

1-Methyl-4-vinylquinolinium tetrafluoroborate:

Trimethyloxonium tetrafluoroborate (1.58 g; 10.7×10⁻³ mol) is added inone portion under an inert atmosphere to a solution of 4-vinylquinoline(0.72 g; 4.64×10⁻³ mol) in a 2.5:1 mixture of acetonitrile/acetone. Thesuspension is left to react for 1 h at 25° C. The solvent is evaporatedunder reduced pressure, and the residue is taken up in 2-propanol (25ml) then stirred for 30 minutes at room temperature. After addition ofethyl ether (25 ml), the suspension is filtered. The desired product isdried under vacuum. Yield=0.93 g (60.4%).

Physical characteristics:

*¹ H NMR (200 MHz, CD₃ COCD₃): 4.30 (s, 3H); 6.25 (d, 1H, J=12.5 Hz);6.65 (d, 1H, J=17.5 Hz); 7.85-8.00 (dd, 1H, J=12.5-17.5 Hz); 8.10 (d,1H, J=7 Hz); 8.35 (m, 2H); 8.57 (d, 1H, J=10 Hz); 8.72 (d, 1H, J=10 Hz);9.35 (d, 1H, J=7 Hz). *MS (FAB) glycerol-NOBA: positive ion M⁺ =170(100) negative ion M⁻ =87 (100).

FAB=fast atom bombardment.

NOBA=meta-nitrobenzyl alcohol.

Microanalysis: Calculated (%): C 56.00; H 4.60; N 5.40 Found (%): C55.56; H 4.35; N 4.12.

III. Application Examples

The operating procedures described hereinafter are examples of theapplication of the assay process, using respectively the reagentsBXT01041 (described in Example 1), BXT01048 (described in Example 3),BXT01049 (described in Example 4) and BXT01050 (described in Example 7),brazilin (commercial product), brazilane (compound of Example 10) and3-O-methylbrazilin (compound of Example 9) to measure SOD activity in anerythrocyte lysate.

EXAMPLE 14 Assay procedure using the reagent BXT01041

A case is used which contains the reagents described below. This case ofreagents must be stored at a temperature of between 0° and 4° C.

Description of the reagents:

R1: 2×10⁻³ M solution of BXT01041 in a 50:50 (v/v) mixture of DMSO/H₂ Ocontaining 0.15M boric acid. (To be stored at between 0° and 4° C.during the measurements; stable for at least one month after preparationunder the abovementioned storage conditions).

R2 : 5×10⁻² M solution of 1,4,6-trimethyl-2-vinylpyridiniumtetrafluoroborate in DMSO containing 25% (w/v) of ethylene glycol. (Tobe stored at between 0° and 4° C. during the measurements; stable for atleast one month after preparation).

E: 62.5/37.5 (v/v) absolute ethanol/chloroform. (To be stored at between0° and 4° C. for the preparation of the sample).

B: 0.05M AMPD/HCl buffer, pH=8.8, containing 0.1 mM DTPA. (Leave toequilibrate in air at 37° C. before the measurements).

Preparation of the sample:

At least 100 μl of total blood are centrifuged at 4° C. and 3000 rpm for10 minutes. The supernatant is removed. The erythrocyte pellet is washedwith 0.9% sodium chloride solution (cooled to between 0° and 4° C.),then centrifugation is carried out at 4° C. and 3000 rpm for 10 minutes.The supernatant is removed. The erythrocyte pellet is taken up and mixedwith 4 times its volume of distilled water, cooled to between 0° and 4°C.

200 μl of the mixture E (cooled to between 0° and 4° C.) are added to125 μl of this erythrocyte lysate diluted to 1/5 on the one hand, and to125 μl of distilled water on the other hand. Each of the solutions ismixed thoroughly and is centrifuged at 4° C. and 3000 rmp for 10minutes. The supernatant thus obtained will be used for the assay,respectively for the sample (with the haemoglobin removed) and for thecontrol.

The supernatants must be stored at between 0° and 4° C. for themeasurements.

Assay procedure:

The measurements are carried out at 37° C. in a spectrophotometer whosemeasurement cells are thermostated.

The reaction mixture corresponding to each assay is prepared at the timeof measurement, according to the proportions given in the table belowfor 1 ml cells.

Each reaction is triggered by the addition of 10 μl of reagent R1. Afterhomogenization of the medium, the change in absorbance is recordedimmediately and followed for 1 min 30 s.

The absorbance is measured at 501 nm in cells with a path length of 1cm, against air.

For each measurement, the reaction medium defined in the table belowmust be incubated for 1 minute at 37° C. before the addition of thereagent R1.

    ______________________________________                                                      CONTROL  SAMPLES                                                ______________________________________                                        R2              20 μl   20 μl                                           Blank sample (*)                                                                              40 μl   --                                                 SAMPLES         --         40 μl                                           B               930 μl  930 μl                                          ______________________________________                                         (*): Distilled water which has undergone the same extraction procedure as     the erythrocyte lysate.                                                  

For each of the measurements, the maximum autoxidation rate, expressedin absorbance units per minute, is determined by evaluating the slope ofthe plot of absorbance over time (at around time t=30s). This slopecorresponds to a phase of autoxidation of the reagent BXT01041 whichmust be linear.

Expression of results:

Given that V_(c) and V_(s) are the rates corresponding, respectively, tothe control and to the sample, the SOD activity of the latter iscalculated using the equation of the straight calibration curve shown inFIG. 1, which represents the changes in the inverse of difference (V_(s)-V_(c)), expressed in min/change in absorbance (min/ΔAbs.) as a functionof the inverse of the concentration of SOD, expressed in ml/U. Thisstraight curve enables the determination of the value on the abscissawhich corresponds to an experimentally measured value on the ordinate.

The inverse of this abscissa is then multiplied by the dilution factorD.

The results are thus expressed in units of SOD activity per ml ofsample, such that:

    Units of SOD activity=1/{0.026×[1/(V.sub.s -V.sub.c)]-0.075}×D

The unit U of SOD activity defined in this method has the singularadvantage of providing an invariable reference, taking into account theabsence of relatively unstable reagents of protein or enzyme types suchas xanthine oxydase.

The use of this unit is highly recommended, but the user may translateit into a weight value of a purified SOD with which he has previouslyconstructed a calibration curve. An estimation by weight of this kindwill be severely affected by the percentage of inactive enzyme presentin the preparation.

The results may also be expressed, for example, in U/ml of total bloodor in U/mg of erythrocyte proteins.

Notes:

The assay procedure may be checked by assaying the SOD activity of anerythrocyte lysate prepared as described above and stored at -70° C. inthe form of aliquots, to serve as control. The SOD activity of suchaliquots is stable for at least 6 months at -70° C.

The assay must be carried out on an aliquot which is thawed at the timeof use.

EXAMPLE 15 Assay procedure using the reagent BXT01048 Identical toExample 14 except that:

In R1, the reagent BXT01041 is replaced by the reagent BXT01048.

The change in absorbance is monitored at 505 nm.

FIG. 2 attached shows the changes in the inverse of the difference(V_(s) -V_(c)), expressed in min/change in absorbance (min/ΔAbs.), as afunction of the inverse of the concentration of SOD, expressed in ml/U,in this example.

    Units of SOD activity=1/{0.028×[1/(V.sub.s -V.sub.c)]-0.077}×D.

EXAMPLE 16 Assay procedure using the reagent BXT01049 Identical toExample 14 except that:

R1 is replaced by R3 which is defined as follows:

2×10⁻³ M solution of BXT01049 in a 50/50 (v/v) mixture of DMSO/H₂ O,containing 0.1M boric acid.

The change in absorbance is monitored at 522 nm for 1 min.

FIG. 3 attached shows the changes in the inverse of the difference(V_(s) -V_(c)), expressed in min/change in absorbance (min/ΔAbs.) as afunction of the inverse of the concentration of SOD, expressed in ml/U,in this example.

    Units of SOD activity=1/{0.023×[1/(V.sub.s -V.sub.c)]-0.063}×D.

EXAMPLE 17 Assay procedure using the reagent BXT01050 Identical toExample 14 except that:

R1 is replaced by R4 which is defined as follows:

2×10⁻³ M solution of BXT01050 in a 50/50 (v/v) mixture of DMSO/H₂₀,containing 0.3M boric acid.

The change in absorbance is monitored at 525 nm for 1 min.

FIG. 4 attached shows the variations in the inverse of the difference(V_(s) -V_(c)), expressed in min/change in absorbance (min/ΔAbs.) as afunction of the inverse of the concentration of SOD, expressed in ml/U,in this example.

    Units of SOD activity=1/{0.070×[1/(V.sub.s -V.sub.c)]-0.102}×D.

EXAMPLE 18 Assay procedure using brazilin

A box is used which is identical to that used in Example 14 except thatin R1 the reagent BXT01041 is replaced by brazilin.

The erythrocyte lysate is obtained as described in Example 14.

200 μl of the mixture E (cooled to between 0° and 4° C.) are added to125 μl of this erythrocyte lysate diluted to 1/5. The combination ismixed and centrifuged at 4° C. and 3000 rpm for 10 minutes. Thesupernatant thus obtained, with the haemoglobin removed, will be usedfor the assay (it must be stored at 4° C. for the measurements).

The assay procedure is the same as in Example 14 except that theabsorbance is measured at 539 nm instead of 501 nm and the "blanksample" is replaced by 50% (v/v) ethanol in distilled water.

Expression of results:

Under the conditions of this assay, one unit of SOD activity (U)corresponds to an increase in the formation of brazilein (autoxidationproduct of brazilin), in relation to the control, of 0.5 μmol.l⁻¹.min⁻¹.

FIG. 5 attached shows the variations in the inverse of the difference(V_(s) -V_(c)), expressed in min/change in absorbance (min/ΔAbs.) as afunction of the inverse of the concentration of SOD, expressed in ml/U,in this example.

    Units of SOD activity=1/{0.034×[1/(V.sub.s -V.sub.c)]-0.065}×D.

EXAMPLE 19 Assay procedure using brazilane

The case of reagents used contains the same reagents as that of Example14, except that the reagent BXT01041 is replaced by brazilane.

Preparation of the sample:

As in Example 18.

Assay procedure:

The procedure as in Example 18 is followed except that each reaction istriggered by the addition of 10 μl of reagent R1 containing brazilaneinstead of brazilin, and the change in the absorbance is monitored for 1min at 520 nm instead of for 1 min 30 s at 539 nm.

Expression of results:

FIG. 6 attached shows the variations in the inverse of the difference(V_(s) -V_(c)), expressed in min/change in absorbance (min/ΔAbs.) as afunction of the inverse of the concentration of SOD, expressed in ml/U,in this example.

    Units of SOD activity=1/{0.069×[1/(V.sub.s -V.sub.c)]-0.077}×D.

EXAMPLE 20 Assay procedure using 3-O-methylbrazilin

The case of reagents used contains the same reagents as in Example 14except that the reagent BX01041 is replaced by 3-O-methylbrazilin.

Preparation of the sample:

The preparation of the sample is identical to that described in Example18.

Assay procedure:

The absorbance is measured at 541 nm. Apart from this the assayprocedure is identical to that described in Example 18.

Expression of results:

The expression of the results is identical to that described in Example18, using a calibration curve specific for 3-O-methylbrazilin.

EXAMPLE 21: Different assay procedure using the reagent BXT01050

A case is used which contains the reagents described below. This casemust be stored at a temperature of between 0° and 4° C.

Description of the reagents:

S1: 0.66×10⁻³ M solution of BXT01050 in 3.2×10⁻² M HCl containing 0.5 mMDTPA and 2.5% of absolute ethanol. (To be stored at between 0° and 4° C.for the measurements; stable for at least one month after preparationunder the abovementioned storage conditions).

S2: 3.33×10⁻² M solution of 1,4,6-trimethyl-2-vinylpyridiniumtetrafluoroborate or trifluoromethylsulfonate in DMSO containing 25%(w/v) of ethylene glycol. (To be stored at between 0° and 4° C. duringthe measurements; stable for at least one month after preparation).

S3: 0.055M AMPD/HCl buffer, pH=8.8 (at 37° C.), containing 3.33 mM boricacid and 0.11 mM DTPA. (Leave to equilibrate in air and at 37° C. beforethe measurements).

Preparation of the sample:

Identical to Example 14.

Assay procedure:

Identical to Example 17 except that:

Each reaction is triggered by the addition of 30 μl of reagent S1.

The reaction medium is prepared at the time of use in the following way:

    ______________________________________                                                      CONTROL  SAMPLE                                                 ______________________________________                                        S2              30 μl   30 μl                                           Blank sample (*)                                                                              40 μl   --                                                 SAMPLE          --         40 μl                                           S3              900 μl  900 μl                                          ______________________________________                                         (*): Distilled water which has undergone the same extraction procedure as     the erythrocyte lysate.                                                  

Expression of results:

The SOD activity of the sample is obtained from the ratio V_(s) /V_(c),whose variation with the concentration of SOD (expressed in units of SODactivity) is given by the following equation:

    V.sub.s /V.sub.c =1+[SOD]/(0.073×[SOD]+0.93).

Under the conditions of this assay, one unit of SOD activity correspondsto a ratio V_(s) /V_(c) of 2 and is called a SOD-525 unit (because thechange in the absorbance is monitored at 525 nm, as in Example 17).

FIG. 7 attached shows the variation in the ratio V_(s) /V_(c) as afunction of the concentration of SOD, expressed in SOD-525 units/ml, inthis example.

    Units of SOD activity=[0.93×(1-V.sub.s /V.sub.c)]/[0.073×(V.sub.s /V.sub.c -1)-1]×D.

REFERENCES

1. FRIDOVICH I. (1986), Biological effects of the superoxide radical;Arch. Biochem. Biophys. 247, 1-11.

2. FRIDOVICH I. (1986a), Superoxide dismutases; Adv. Enzymol. 58, 61-97.

3. FRIDOVICH I. (1989), Superoxide dismutases; an adaptation to aparamagnetic gas; J. Biol. Chem. 264, 7761-7764.

4. KARLSSON K. and MARKLUND S. L. (1988), Extracellular superoxidedismutase in the vascular system of mammals; Biochem. J., 255, 223-228.

5. CZAPSKI G. and GOLDSTEIN S. (1988), The ubiquiness both of superoxidetoxicity and of the protective role of superoxide dismutase; in: OxygenRadicals in Biology and Medicine, SIMIC M. G., TAYLOR K. A., WARD J. F.and VON SONNTAG C. Ed., Plenum Press, New York and London, 43-46.

6. FRIDOVICH I. (1982b), Measuring the activity of superoxidedismutases: an embarrassment of riches; in: Superoxide dismutase Vol. I,OBERLEY L. W. Ed., CRC Press, Boca Raton, Fla., 69-77.

7. FLOHE L. and OTTING F. (1984), Superoxide dismutase assays; Meth.Enzymol., 105, 93-104.

8. BANNISTER J. V. and CALABRESE (1987), Assays for superoxidedismutase; in: Methods of Biochemical Analysis, Vol. 32, John Wiley &Sons, 279-311.

9. FLOHE L., BECKER R., BRIGELIUS R., LENGFELDER E. and OTTING F.(1989), Convenient assays for superoxide dismutase; in: CRC Handbook;Free Radicals and Antioxidants in Biomedicine, Vol. III, MIQUEL J.,WEBER H. and QUINTANILHA A. Eds., CRC Press, Boca Raton, Fla., 287-293.

10. GOLDSTEIN S., MICHEL C., BORS W., SARAN M. and CZAPSKI G. (1988), Acritical reevaluation of some assay methods for superoxide dismutaseactivity; Free Rad. Biol. Med., 4, 295-303.

11. MARKLUND S. L. (1976), Spectrophotometric study of spontaneousdisproportionation of superoxide anion radical and sensitive directassay for superoxide dismutase; J. Biol. Chem., 251, 7504-7507.

12. MARKLUND S. L. (1985), Quantitation of superoxide dismutase: Directassay with potassium superoxide; in: CRC Handbook of Methods for OxygenRadical Research, GREENWALD R. A. Ed., CRC Press, Boca Raton, Fla.,249-255.

13. MARTIN J. P., DAILEY M. and SUGARMAN E. (1987), Negative andpositive assays of superoxide dismutase based on haematoxylinautoxidation; Arch. Biochem. Biophys., 255, 329-336.

14. MARTIN J. P. (1990), Assays for superoxide dismutase based onautoxidation of haematoxylin; Meth. Enzymol., 186, 220-227.

15. TRUCE, KREIDER and BRAND (1971), Org. React., 18, 99-215.

16. COMINS D. L., STROUD E. D. and HERRICK J. J. (1984), Regioselectivealkylation of Grignard reagents to the 1-phenoxycarbonyl salts of alkylnicotinates; Heterocycles, 22 (1), 151-157.

We claim:
 1. A process for the assay of superoxide dismutase (SOD)activity in a liquid medium, which uses activation of a reagent by SODactivity comprising the steps of:1) adding a compound of general formulaI ##STR7## in which: either: n=1 or 2, R¹ =--OR⁴ or --NR⁵ R⁶, R²=hydrogen, --OR⁴, C₁₋₆ alkyl, --CH₂ or --CH₂ --CH₂ --, to form a ring bylinking with the phenyl substituent, in the meta position in relation toR¹ ; and R³ =hydrogen, C₁₋₆ alkyl or --OR⁴ ; with the proviso that R² isdifferent from --OR⁴,where R⁴ =hydrogen or C₁₋₆ alkyl; R⁵ =hydrogen,C₁₋₆ alkyl, --CH₂ COOH, --C₆ H₅ SO₃ H; and R⁶ =hydrogen, C₁₋₆ alkyl or--CH₂ COOH; or: n=1, R¹ =--OR⁴ ; R⁴ being defined as above, R² =--CH₂--O--, to form a ring by linkage of the oxygen atom with the phenylsubstituent, in the meta position in relation to R¹, and R³ =hydrogen or--OR⁴ ; R⁴ being defined as above, to the medium; and 2)spectrophotometric measurement of the autoxidation rate of said compoundof general formula I in the absence and presence of the sample to beassayed.
 2. The process for the assay of superoxide dismutase (SOD)activity in a liquid medium containing one or more mercaptans, whichprocess utilizes activation of the autoxidation of a reagent by SODactivity wherein the said reagent of general formula defined in claim1,further comprising adding a quantity which is capable of totallyscavenging the said mercaptans by S-alkylation, of at least one compoundconforming to the general formula II: ##STR8## in which: Z¹ =C₁₋₆ alkyl,benzyl, p-nitrobenzyl, phenyl, o,p-dinitrophenyl, --CH₂ --COOH or --CH₂--CH₂ --COOH; Z⁴ =hydrogen and Z⁵ =hydrogen or C₁₋₆ alkyl, or Z⁴ and Z⁵form together with the two intermediate carbon atoms; a phenyl ring;X=halide, sulfonate, fluorosulfonate, phosphonate, tetrafluoroborate ortosylate; and either Z² =vinyl, and Z³ =hydrogen or C₁₋₆ alkyl, or Z³=vinyl, and Z² =hydrogen or C₁₋₆ alkyl, andwherein said process residesin the spectrophotometric measurement of the autoxidation rate of saidcompound of general formula I in the absence and presence of the sampleto be assayed.
 3. The process according to claim 2, wherein in saidgeneral formula II, X=trifluoromethylsulphonate.
 4. A process for theassay of superoxide dismutase (SOD) activity in a liquid sample,comprising the steps of:1) adding a compound of general formula I##STR9## in which: either: n=1 or 2, R¹ =--OR⁴ or --NR⁵ R⁶, R²=hydrogen, --OR⁴, C₁₋₆ alkyl, --CH₂ or --CH₂ --CH₂ --, to form a ring bylinking with the phenyl substituent, in the meta position in relation toR¹ ; and R³ =hydrogen, C₁₋₆ alkyl or --OR⁴ ; provided that R² isdifferent from --OR⁴, where R⁴ =hydrogen or C₁₋₆ alkyl; R⁵ =hydrogen,C₁₋₆ alkyl, --CH₂ COOH, --C₆ H₅ SO₃ H; and R⁶ =hydrogen, C₁₋₆ alkyl or--CH₂ COOH; or: n=1, R¹ =--OR⁴ (R⁴ being defined as above), R² =--CH₂--O--, to form a ring by linkage of the oxygen atom with the phenylsubstituent, in the meta position in relation to R¹ ; and R³ =hydrogenor --OR⁴ ; R⁴ being defined as above;to the sample; 2) contacting thesample with the compound in a reaction medium which is buffered to a pHvalue of from 8.0 to 9.0, triggering the reaction by the addition of analiquot of a stock solution of the compound; 3) determining the maximumrate of autoxidation, V_(s) of the compound in the presence of thesample, by means of the change in the absorbance as a function of time,at the wavelength which characterizes the appearance of the autoxidationproduct of the compound; 4) determining, under the same conditions, themaximum autoxidation rate, V_(c) of the same compound in the absence ofthe sample; and 5) determining under the same conditions, the maximumautoxidation rates for standard samples of known concentration in SODand plotting a calibration curve setting forth the reverse of theconcentration in SOD as the abscissa with respect to the reverse of thedifference of the maximum autoxidation rates 1/V_(s) -V_(c) obtained asthe ordinate; and 6) determining the SOD activity by means of thereverse of the difference of the maximum autoxidation rates, V_(s) andV_(c) obtained from steps (3) and (4) by using the calibration curve. 5.The process according to claim 1, wherein said process comprises theaddition of a boron derivative, to modulate the autoxidation rate of thecompound of general formula I.
 6. The process according to claim 4,wherein the determination of the maximum rate of autoxidation V_(s)under the conditions of activation of the autoxidation of the compoundof general formula I is combined with the determination of the maximumrate of the autoxidation of the compound of general formula I underconditions in which this autoxidation is inhibited, in a pH range from7.2 to 7.8.
 7. The process according to claim 1, wherein the compound ofgeneral formula I is5,6,6a,11b-tetrahydro-3,9,10-trihydroxybenzo[c]fluorene.
 8. Kit for theimplementation of the process according to claim 1 which comprises areagent compound of the general formula I and a container for thereagent.
 9. The kit according to claim 8, further comprising one or moremercaptan-scavenging compounds of general formula II.
 10. The kitaccording to claim 9, further comprising:a compound of general formula Iin acidic solution or in a powder, one or more mercaptan-scavengingcompounds of general formula II, in solution or as a powder, and abuffer based on AMPD, which buffers in the pH range from 8.0 to 9.0. 11.A compound of general formula I ##STR10## in which: either:n=1 or 2, R¹=--OR⁴ or --NR⁵ R⁶, R² =hydrogen, --OR⁴, C₁₋₆ alkyl, --CH₂ or --CH₂--CH₂ --, to form a ring by linking with the phenyl substituent, in themeta position in relation to R¹ ; and R³ =hydrogen, C₁₋₆ alkyl or --OR⁴; provided that R² is different from --OR⁴,where R⁴ =hydrogen or C₁₋₆alkyl; R⁵ =hydrogen, C₁₋₆ alkyl, --CH₂ COOH, --C₆ H₅ SO₃ H; and R⁶=hydrogen, C₁₋₆ alkyl or --CH₂ COOH;or: n=1, R¹ =--OR⁴ ; R⁴ beingdefined as above, R² =--CH₂ --O--, to form a ring by linkage of theoxygen atom with the phenyl substituent, in the meta position inrelation to R¹ ; and R³ =hydrogen or --OR⁴ ; R⁴ being defined asabove;with the proviso that when n=1 and R² =--CH₂ O--, R¹ and R³ maynot simultaneously represent --OR⁴ where R⁴ =hydrogen.
 12. A compound ofgeneral formula I ##STR11## in which: n=1 or2;R¹ =--OR⁴ or --NR⁵ R⁶, R²=hydrogen, --OR⁴, C₁₋₆ alkyl --CH₂ or --CH₂ --CH₂ --, to form a ring bylinking with the phenyl substituent, in the meta position in relation toR¹ ; and R³ =hydrogen, C₁₋₆ alkyl or --OR⁴ ; with the proviso that R² isdifferent from --OR4,where R⁴ =hydrogen or C₁₋₆ alkyl; R⁵ =hydrogen,C₁₋₆ alkyl, --CH₂ COOH, --C₆ H₅ SO₃ H; and R⁶ =hydrogen, C₁₋₆ alkyl or--CH₂ COOH.
 13. A process for preparing the compound of general formulaI ##STR12## in which: n=1 or 2;R¹ =--OR⁴ or --NR⁵ R⁶, R² =hydrogen,--OR⁴, C₁₋₆ alkyl, --CH₂ or --CH₂ --CH₂ --, to form a ring by linkingwith the phenyl substituent, in the meta position in relation to R¹ ;and R³ =hydrogen, C₁₋₆ alkyl or --OR⁴ ; provided that R² is differentfrom --OR⁴,where R⁴ =hydrogen or C₁₋₆ alkyl; R⁵ =hydrogen, C₁₋₆ alkyl,--CH₂ COOH, --C₆ H₅ SO₃ H; and R⁶ =hydrogen, C₁₋₆ alkyl or --CH₂COOH;or: n=1, R¹ =--OR⁴ ; R⁴ being defined as above, R² =--CH₂ --O--, toform a ring by linkage of the oxygen atom with the phenyl substituent,in the meta position in relation to R¹ ; and R³ =hydrogen or --OR⁴ ; R⁴being defined as above;in which n=1, R⁴ =OH, R² =CH₂ --O-- and R³=hydrogen, or brazilane, which comprises the steps of: (1) protectingbrazilin on its phenolic hydroxyls, (2) transforming the alcoholichydroxyl into a group which enables its removal, and (3) reducing,deprotecting and isolating the resulting product.
 14. A process for theassay of superoxide dismutase (SOD) activity in a liquid sample,comprising the steps of:1) adding a compound of general formula I##STR13## in which: either: n=1 or 2, R¹ =--OR⁴ or --NR⁵ R⁶, R²=hydrogen, --OR⁴, C₁₋₆ alkyl or --CH₂ --CH₂ --, to form a ring bylinking with the phenyl substituent, in the meta position in relation toR¹ ; and R³ =hydrogen, C₁₋₆ alkyl or --OR⁴ ; provided that R² isdifferent form --OR⁴ ; where R⁴ =hydrogen or C₁₋₆ alkyl; R⁵ =hydrogen,C₁₋₆ alkyl, --CH₂ COOH, --C₆ H₅ SO₃ H; and R⁶ =hydrogen, C₁₋₆ alkyl or--CH₂ COOH; or: n=1, R¹ =--OR⁴ ; R⁴ being defined as above, R² =--CH₂--O--, to form a ring by linkage of the oxygen atom with the phenylsubstituent, in the meta position in relation to R1; and R³ =hydrogen or--OR⁴ ; R⁴ being defined as above, to the sample; 2) contacting thesample with the compound in a reaction medium which is buffered to a pHvalue of from 8.0 to 9.0, triggering the reaction by the addition of analiquot of a stock solution of the compound; 3) determining the maximumrate of autoxidation, V_(s) of the compound in the presence of thesample, by means of the change in the absorbance as a function of time,at the wavelength which characterizes the appearance of the autoxidationproduct of the compound; 4) determining, under the same conditions, themaximum autoxidation rate, V_(c) of the same compound in the absence ofthe sample; 5) determining under the same conditions, the maximumautoxidation rates for standard samples of known concentration in SODand plotting a calibration curve, setting forth the SOD concentration asthe abscissa and the ratio of V_(s) and V_(c) as the ordinate; and 6)determining the SOD activity of the liquid sample by means of the ratio,V_(s) and V_(c) obtained with the liquid sample under steps (3) and (4)by using said calibration curve.
 15. The process according to claim 1wherein the liquid medium is a biological medium from an aerobicorganism and human organism.
 16. The process according to claim 5wherein the boron derivative is boric acid.
 17. The process according toclaim 15 wherein the biological medium is blood.
 18. The processaccording to claim 15 wherein the biological medium is selected from agroup consisting of blood plasma, erythrocytes, blood platelets,leucocytes, synovial fluid, cerebrospinal fluid, urine and tissueextract.